November 06, 2024

Genetics and Genomic Breeding

Grapevine pangenome facilitates trait genetics and genomic breeding


Grapevine breeding is hindered by a limited understanding of the genetic basis of complex agronomic traits. This study constructs a graph-based pangenome reference (Grapepan v.1.0) from 18 newly generated phased telomere-to-telomere assemblies and 11 published assemblies. Using Grapepan v.1.0, we build a variation map with 9,105,787 short variations and 236,449 structural variations (SVs) from the resequencing data of 466 grapevine cultivars. Integrating SVs into a genome-wide association study, we map 148 quantitative trait loci for 29 agronomic traits (50.7% newly identified), with 12 traits significantly contributed by SVs. The estimated heritability improves by 22.78% on average when including SVs. We discovered quantitative trait locus regions under divergent artificial selection in metabolism and berry development between wine and table grapes, respectively. Moreover, significant genetic correlations were detected among the 29 traits. Under a polygenic model, we conducted genomic predictions for each trait. In general, our study facilitates the breeding of superior cultivars via the genomic selection of multiple traits.

Main

The cultivated grapevine (Vitis vinifera ssp. vinifera L.) is an economically important perennial fruit crop that is grown widely for winemaking and fresh fruit in ~94 countries. Previous studies have suggested that grapevine originated from a single domestication event in the Black and Caspian Sea regions more than 10,000 years ago, which subsequently spread across the northern hemisphere with gene flow from local wild populations. However, other studies have suggested the potential for multiple domestication events. Since domestication, grapevine cultivars have accumulated deleterious genomic variants, including single-nucleotide polymorphisms (SNPs) and SVs, in a heterozygous state, resulting in strong inbreeding depression. Recent studies have highlighted the potential contribution of hidden genomic variants, including SVs to phenotypes, but the quantitative genetic basis of complex agronomic traits in grapevine has rarely been investigated at the genome scale.

Long-read sequencing technologies have revealed the prevalence of SVs in plant genomes. It is increasingly evident that SVs are more likely than SNPs to influence the phenotype of domestication traits. At the population level, SVs tend to occur at low frequencies, reflecting negative selection signals. Furthermore, the frequency of SVs may be related to their recent origin. For example, recent transposable element (TE) activity can generate new SVs that are initially present in only one individual or lineage. In part because of their low population frequencies, SVs are typically in low linkage disequilibrium (LD) with SNPs. One practical implication of low LD is that SVs may encompass substantial missing heritability for quantitative traits. Consistent with this viewpoint, the addition of SVs to population and quantitative genetic analyses has yielded new insights into local adaptation and agronomic traits.

Grapevine genomes are highly heterozygous, partly because of the accumulation of genetic variation during clonal propagation, which has been carried out for thousands of years. For example, the genomes of diploid Chardonnay and Cabernet Sauvignon contain more than 10% heterozygous sites including SNPs, insertion–deletions (indels) and SVs. Although the commonly used reference genome from PN40024 was highly homozygous after nine generations of selfing, it is missing >10% of genes compared with heterozygous cultivars. Across cultivars, only ~7% of the genes are shared, whereas ~8% are unique to each individual. The high level of variability in grapevine merits the construction of a pangenome reference that incorporates presence–absence variation, improves the detection of genomic variants, including SV, and reduces reference biases.

Here we assembled 18 haplotype-resolved telomere-to-telomere (T2T) assemblies representing eight diploid grapevine cultivars and one diploid wild grape. We then constructed a graph-based pangenome, which we call Grapepan v.1.0, using these new assemblies and 11 previously published chromosomal assemblies. These genotypes represent the global genetic diversity of grapes. Using Grapepan v.1.0, we built a variation map that includes SNPs, indels (2 bp ≤ indel < 50 bp) and SVs (≥50 bp) across a larger sample of 466 accessions, including 324 that were newly sequenced. We utilized this variation map in a genome-wide association study (GWAS) and the genomic prediction of 29 complex agronomic traits. This exercise identified quantitative trait loci (QTLs) for these agronomic traits, provided unique insights into the contribution of SVs to quantitative genetic variation and demonstrated the feasibility of breeding superior cultivars via genomic selection for multiple traits. The pangenome reference (Grapepan v.1.0), variation map, QTLs and our genomic selection models facilitate genomic breeding of grapevine.

Discussion

Accelerating the innovation of grape varieties is urgently required to adapt to future planting, rapidly changing market demands and climate change. Grape breeding exhibits a degree of reliance on older varieties; in particular, clonal reproduction allows the preservation of genotypes over extended periods, some of which are older than 900 years. Advances in grapevine breeding lag far behind those made in annual cereal crops because of their long generation times (~3 years on average), high deleterious burden that leads to inbreeding and/or hybrid depression, high genomic heterozygosity, inefficient genetic transformations and limited knowledge about the genetic basis of complex agronomic traits.

Progress in understanding the complexity of the grapevine over the past two decades, from phenotypic characterization to marker identification and association analysis, has greatly benefited breeding efforts. Early breeding emphasized correlation analysis between phenotypic traits and low-density genetic markers, and selected phenotypic traits through marker-assisted selection. Using association analyses, researchers have linked specific genetic variations to desirable phenotypic traits, providing breeders with valuable tools for the targeted selection of multiple phenotypes, including berry size, color and sugar content. These efforts have led to significant contributions such as the development of seedless grape varieties and the enhancement of disease resistance in grapevines. However, limitations inherent in detecting variations within a single reference genome hinder the identification of crucial variants associated with breeding traits and a comprehensive analysis of agronomic trait inheritance.

Advanced pangenome-based approaches underscore broader efforts aimed at discovering genetic variants in crop breeding. Recent research has focused on North American wild grapevines and has established a nonreference pangenome inclusive of nine wild accessions. Their sequencing encompasses the diversity of wild grapevine species, aiming to integrate resistance variants from wild species for use in rootstock improvement. By contrast, our pangenome (Grapepan v.1.0) focuses on discovering variants associated with agronomic traits in domesticated grapevines. We selected representative cultivated varieties to construct the pangenome. We also included table grape varieties to expand diversity across grape populations with different uses. Therefore, our pangenome may contain more advantageous genotypes related to domesticated traits, thus directly serving breeding programs. We utilized a graph-based approach in which any variant is integrated as a node within the pangenome reference. Indeed, the most significant enhancement of the pangenome lies in the discovery of SVs. The number of newly discovered SNPs differs only slightly compared with alignment with a single reference genome or previous pangenome versions. Thus, our grape pangenome places greater emphasis on uncovering traits associated with SVs and revealing their inheritance patterns.

In Grapepan v.1.0, SVs often associated with repetitive sequences and TEs, suggesting that TE-mediated events are an important evolutionary force. The low frequency of SVs in the grapevine genome can be attributed to recent TE activity and the evolutionary constraints imposed by natural selection. This poses challenges in precisely controlling the breeding process when relying solely on SNP for trait selection. This challenge is exacerbated by the incomplete capture of heritability for multiple traits, particularly from SVs, which might be related to two factors. First, LD decay can influence the resolution of genetic mapping and the identification of causal variants. Second, SVs are often found to generate and explain a greater proportion of phenotypic variation in numerous traits compared with SNPs. The rarity of SVs also makes it difficult to accurately estimate their frequency and effect size within a population. Consequently, the statistical power to detect associations involving rare SVs is lower than that for SNPs. In addition, SVs are larger relative to SNPs and can engender more immediate functional consequences, such as perturbations in gene dosage or the disruption of critical gene regulatory elements. For example, SVs contribute the largest share of heritability for approximately half of the molecular traits in tomatoes, the identification of SVs based on pangenome has greatly increased estimates of the heritability of metabolic traits. In foxtail millet, the precision of 73.9% of traits with both SNP and SV markers increased by between 0.04% and 12.67% compared with SNP-only markers. Fruit color serves as a key trait in grape breeding, renowned for its association with SV determination. We confirmed the higher heritability in fruit color contributed by SV and emphasized the power and accuracy of SV-based GWAS and genomic selection. We have found that the inheritance of an isoamylase gene associated with a 5.6-kb deletion explained 6.23% of the variance in SSC. Collectively, a deep understanding of SVs based on the pangenome will greatly improve the efficiency of SV-associated analysis for grapevine breeding.

Results

The graph pangenome reference for grapevine (Grapepan v.1.0)

HiFi reads, Hi-C reads and ultra-long nanopore reads were collected for nine representative diploid samples, including one accession of Vitis retordii, a wild species endemic to Asia, and eight grapevine cultivars (seven table grapes and one wine grape). The nine samples resulted in 18 haplotypes that reached T2T-level assembly after gap filling. Genome sizes ranged from 479.15 to 539.30 Mb . The quality of haplotype assembly was confirmed by high contiguity (>99.9%), minimal switching error (<0.05%) and low Hamming error (<2.83%). Benchmarking universal single-copy orthologs evaluation indicated an average completeness of 98.4% for these haplotypes (range 98.07% to 98.64%). We used the same pipeline to annotate all haplotypes and to ensure consistent results. Across the 18 haplotypes, the number of protein-coding genes ranged from 34,536 to 38,526, and the TE sequence length per haplotype ranged from 263.86 Mb (54.68%) to 312.10 Mb (59.03%). In addition, we identified centromere and telomere sequences in all assemblies. Consistent with previous studies, the predominant repeat unit of the centromere was 107 bp long. Overall, these 18 assembled haplotypes and their annotations represent one of the highest-quality grapevine genomic datasets generated to date.

gene editing, genetic markers, genome sequencing, CRISPR technology, genetic mapping, molecular breeding, quantitative trait loci (QTL), genetic diversity, gene expression profiling, functional genomics, genome-wide association studies (GWAS), transgenic crops, epigenetics, genetic resistance, hybrid vigor, molecular markers, phenotypic variation, DNA barcoding, polygenic traits, marker-assisted selection,

#Genetics, #GenomicBreeding, #GeneEditing, #GenomeMapping, #CRISPR, #GWAS, #MolecularBreeding, #Epigenetics, #GenomicSelection, #GeneticDiversity, #MarkerAssistedSelection, #FunctionalGenomics, #Phenotyping, #TransgenicCrops, #QTLMapping, #DNASequencing, #HybridVigor, #GeneExpression, #PlantBreeding, #GeneticImprovement

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    November 05, 2024

    Mandatory Genetic Testing

    Mandatory genetic testing in sport ‘not justified, ethical or viable’, say academics


    Mandatory testing of sex chromosomes in sport is neither justified, ethical nor viable, a group of academics say.

    Safety and fairness in female sport was one of the most prominent topics at this summer’s Olympic Games, where boxers Imane Khelif and Lin Yu Ting took gold in the women’s welterweight and featherweight categories respectively.

    The International Boxing Association had disqualified the fighters from its World Championships last year for allegedly failing gender eligibility tests, though the governing body failed to provide any concrete evidence in support of this at a press conference during the Games.

    The IBA had been stripped of recognition by the International Olympic Committee (IOC) over governance failures and therefore it was the IOC which ran – and set the entry criteria for – the Olympic boxing competitions in Paris.

    During the Games an editorial by a group of scientists in the Scandinavian Journal of Medicine and Science in Sports (SJMSS) proposed the introduction of sex chromosome testing amid what it described as a “growing concern” over the participation in female sport of athletes with an XY difference of sexual development (XY DSD).

    However, another group of experts has now questioned the proposed testing regime in an editorial in the same journal, published on Monday.

    The group, which includes Professor Alun Williams from Manchester Metropolitan University Institute of Sport, first of all highlighted the lack of direct evidence demonstrating a performance advantage for athletes with XY DSD.

    Secondly, they believe the first editorial’s call for “early” testing at the sub-elite level must include minors if it is to achieve its aim. They say the concerns which led to genetic testing being abandoned in 1999 remain, “and are amplified by the vastly increased number of younger athletes” it is proposed would be tested under the new regime.

    “The editorial gives the impression that such tests are straightforward – ‘individual consent, confidentiality, and dignity… simple cheek swab… standard medical care’, but these assurances ignore the enormous problems such a testing regime would generate,” the group wrote.

    They argue that under the proposal for mandatory genetic testing for sport eligibility, “young athletes would not be presented with a genuine choice” and some would be subjected to invasive examination by gynaecologists.

    “Consent is only a coercive offer: comply with the test or never participate in competitive women’s or girls’ sport, even at sub-elite level,” they wrote.

    They also question who would pay for and produce the “worldwide army of counselling expertise” required to operate an ethically responsible genetic testing programme.

    “For those undergoing follow-up clinical examination and genome sequencing….how would the devastation of young athletes’ personal identity and self-esteem, and the alarm caused to their families, be managed?” they wrote.

    “The resultant duty of care of these athletes will fall to the sport federations mandating such assessments, without any realistic prospect of being fulfilled.”

    The new editorial concludes: “Broad discussion is required to develop more appropriate regulations. However, the proposed mandatory testing of all young women and girls in sport is not justified by scientific evidence, has limited ethical defensibility, and is not an operationally viable proposition.”

    Khelif filed a legal complaint with the French authorities over the online abuse and harassment she was subjected to during the Olympic Games.

    A response to the Williams group’s editorial was also published in the SJMSS on Monday.

    The response said the Williams group appeared to have applied a “no presumption of advantage” principle to XY DSD athletes.

    They argue that evidence shows athletes with certain XY DSDs have: male genitals and testosterone levels within the male range, sensitivity to male-range testosterone which makes the body more masculine, a prevalence of 140 times higher in female sports compared to the general population, and reduced performance where testosterone is suppressed.

    “It thus follows that athletes with these XY DSDs hold male performance advantages,” the response from a group including sports scientist Ross Tucker stated.

    They also rejected the idea that testing would need to be done on minors.

    “Rather, we believe that eligibility screening should occur early enough in an athlete’s career to protect their privacy and dignity and avoid the ethical failures of the past,” they wrote.

    Their response concluded: “We believe that a broader screening process with follow-up examinations in rare cases is scientifically sound, ethically justifiable and operationally feasible.”

    Genetic testing, mandatory screening, genetic privacy, consent, genetic discrimination, health insurance, ethical concerns, privacy rights, genetic data, genetic counseling, personalized medicine, genetic predisposition, risk assessment, disease prevention, DNA profiling, genomic data security, bioethics, healthcare policy, public health, genetic diversity,

    #GeneticTesting, #MandatoryScreening, #GeneticPrivacy, #InformedConsent, #GeneticDiscrimination, #HealthInsurance, #EthicsInGenomics, #PrivacyRights, #GeneticData, #GeneticCounseling, #PersonalizedMedicine, #RiskAssessment, #PreventionThroughGenetics, #DNAProfiling, #GenomicSecurity, #Bioethics, #HealthcarePolicy, #PublicHealth, #GeneticDiversity, #FutureOfHealthcare

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    November 04, 2024

    Chemotherapy

    Chemotherapy Could Harm Or Kill You If You Have This Genetic Finding



    Chemotherapy has been used for decades to treat patients with cancer. One common type of chemotherapy - called fluorouracil (5-FU) when given by IV, or capecitabine (Xeloda or CAPE) when given by pill – can be toxic or fatal to a small percentage of people who carry a genetic change in a gene called DPYD. The prevalence of this genetic finding varies in different populations. Data suggest that for people who carry one DPYD variant, 2-3% will die of a treatment-related fatality if they take one of these medications (25x higher than the risk of an average person receiving the same drugs at an average dose).

    Why? We all carry 2 copies of a gene called DPYD that makes a chemical (DPD) that helps our bodies break down and get rid of these drugs after our body uses them. This process helps prevent the patient from developing toxic or fatal side effects from this chemo. But, up to 8% of people carry one variant in this gene that causes a partial deficiency of DPD. This can double their exposure to the toxic effects of these medications at standard doses. Two in 1000 patients carry two variants that result in a complete absence of DPD function – in such patients, exposure to 5-FU is often fatal.


    If a patient develops toxicity to 5-FU or capecitabine, and it is recognized immediately, an antidote called uridine triacetate can be administered (at a total cost of >$180,000 USD for medication and care). But this process must occur quickly, so patients experiencing severe side effects must contact their providers without delay. An example used to illustrate such side effects is that of “an active and independent patient who starts a planned 14-day cycle of capecitabine and cannot walk to the bathroom independently by day 10.” Patients who have two variants in the gene will likely be much sicker, much faster.

    Drug authorities in the UK and European Union have recommended pre-treatment DPYD testing since 2020. If patients there carry one copy of the variant (genetically intermediate metabolizers of the drug), they are first given half of the standard dose to see how they do. If the patients do okay when they take the drug, doctors can raise the dose. If the patient carry two variants in the gene (a complete absence of DPD), other drugs are recommended instead if possible, or a quarter of the usual starting dose is used.

    Only recently the United States FDA issued warnings about this issue. In 2024, the agency issued a statement that physicians should “consider testing” before prescribing 5-FU and capecitabine and “must inform patient of availability of DPYD testing and the implications of testing.” The FDA stopped short of recommending that clinicians order genetic testing for DPYD on their patients before prescribing 5-FU or capecitabine. These rather loose recommendations, and the fact that highly influential cancer organizations have not supported testing have contributed to the problem. Only 3% of oncologists in the United States order such testing before giving patients these drugs. Implementing pre-treatment DPYD genetic testing can be challenging, but some major healthcare systems have done so successfully. Most U.S. insurers, both private and public, will cover genetic testing for DPYD (which typically costs less than $450), and studies have shown that this testing is cost-effective.

    Different gene variants are found in people from various ethnic backgrounds. A Canadian physician, Dr. Anil Kapoor, who was diagnosed with colon cancer and tested negative for four common DPYDvariants died after one dose of 5-FU. Why? Those four common variants are found most often in people of European ancestry and Dr. Kapoor was of South Asian ancestry. Testing performed after his death showed that Dr. Kapoor indeed carried another variant in this gene. The Association of Molecular Pathology (AMP) recently published guidelines for which DPYD variants should be included on panels before these drugs are administered to ensure that tests better account for ethnic diversity.

    · Genetic testing is changing rapidly. Most clinicians cannot keep up with the genetic factors that play into drug reactions (a specialty called pharmacogenetics) in addition to their own areas of work.

    o Pharmacists and certified genetic counselors who specialize in this area can help. But, remarkably, these graduate-level health care professionals are not recognized as ‘healthcare providers’ by Medicare and Medicaid, and therefore many insurance companies. Without this recognition by our federal government it is difficult for these providers to get reimbursed for their services. Therefore, they are not utilized in patient care as often as they could and should be. However, “the downstream cost benefits (fewer adverse events, fewer hospitalizations, decreased length of stay in the hospital, patient satisfaction, etc.) can easily outweigh the cost of having a dedicated full-time employee” to support such services, explains Jai Patel, PharmD, CPP, Director, Cancer Pharmacology & Pharmacogenomics at Atrium Health Levine Cancer Institute in Charlotte, NC.

    o In addition to offering DPYD testing, genetic panels are available that can help assess patients’ risk for adverse reactions to other chemotherapies and medications, such as those used to treat pain, depression, nausea, and acid-reflux. While many people experience these conditions, they are even more common in people with cancer. Therefore, pharmacogenetic testing may be very helpful in guiding the selection or dosing of other medications, with one study showing that the use of such panels in cancer care reduced adverse events by more than 50%.

    o Some clinicians fear that genetic testing will delay treatment or feel that they can manage the symptoms if they occur, and do not wish to give lower doses of these medications. However, there are now data to suggest that lowering doses in these patients does not have a negative impact on their survival.

    o Interpreting genetic testing panels can be challenging. Some clinicians fear that if they order this testing, they are responsible for interpreting the results and using it correctly, and worry that this creates liability for them and their health system. But some health systems, like OSHU, have been sued because they didn’t offer this testing and a patient died.

    Clinical decision support tools for pharmacogenetics are available. They help the clinician by providing guidance on when genetic testing should be used, and what drug or dose to use to maximize safety and effectiveness. Kristine Ashcraft, President and Founder of YouScript, has devoted the last two decades to creating such tools. “Most providers have little exposure to genetics in their medical training and are understandably fearful of incorporating something they don’t understand into clinical decision-making. But when provided with tools to guide them, the majority of healthcare providers say this becomes easy to navigate.“

    But many health systems don’t use these tools, or the tools are not easily and readily available in electronic medical records (EMR), or pharmacy systems. One pharmacist spent a year building his own digital tools in the EMR called Epic. Those tools will only help his health system and will require updates as the field changes. Many of the large multi-billion dollar EMR companies make it difficult for smaller companies to integrate their own digital tools, often requiring expensive and time-consuming hospital-by-hospital integration and charging hefty fees to be included in their networks. The FDA and our federal government should examine these power structures and how they influence the everyday practice of medicine.

    #ChemotherapyRisk, #DPDDeficiency, #Pharmacogenomics, #CancerTreatment, #GeneticTesting, #OncologySafety, #PersonalizedMedicine, #DrugMetabolism, #ToxicityPrevention, #CancerCare, #5Fluorouracil, #Capecitabine, #GeneticScreening, #PrecisionMedicine, #OncologyResearch, #MedicalGenetics, #PatientSafety, #AdverseReactions, #CancerAwareness, #TumorTherapy

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    November 02, 2024

    Genetic Science

    3X4 Genetics and Rupa Health Partnership Aims to Bridge the Gap Between Genetic Science and Clinical Practice

    SEATTLE, Oct. 31, 2024 /PRNewswire/ -- 3X4 Genetics, a global leader in genetic testing, is proud to announce an exciting new development in their partnership with Rupa Health, a renowned provider of integrative healthcare solutions. The partnership provides healthcare professionals and patients the ability to harness the power of genetic information for more personalized and holistic care.

    Throughout this strategic alliance, 3X4 Genetics and Rupa Health aim to bridge the gap between genetic science and clinical practice. By combining 3X4 Genetics' technologically advanced genomic insights with Rupa Health's innovative diagnostic platform, healthcare practitioners will be better equipped to deliver customized treatment plans tailored to each patient's unique needs. The collaboration will allow doctors and healthcare professionals to access genetic data alongside comprehensive testing services, improving health outcomes, and empowering patients to make more informed decisions.

    "Our collaboration with Rupa Health allows us to push the boundaries of what personalized healthcare can achieve. At 3X4 Genetics, we've always believed in the potential of genomic science to transform health, and with Rupa Health's expertise in diagnostics and patient care, we can now accelerate that vision. Together, we are building a future where genetic insights lead to more precise and proactive care for all," says Dr. Yael Joffe, the Chief Scientific Officer and founder of 3X4 Genetics.

    By connecting their software, both companies ensure that healthcare remains adaptive, dynamic, and patient-centered."Having this report on Rupa is an incredible resource for practitioners to better support their patients with complex genetic outcomes," shares Tara Viswanathan, CEO and founder of Rupa Health.

    By emphasizing the power of genetic testing to guide more effective, personalized health interventions, the partnership between 3X4 Genetics and Rupa Health is set to redefine how healthcare professionals approach patient care.

    About 3X4 Genetics

    3X4 Genetics is at the forefront of genetic testing and insights, empowering healthcare practitioners and individuals to harness the power of genetics to optimize health and well-being. With a focus on integrative and personalized healthcare, 3X4 Genetics uses cutting-edge genomic science to provide actionable health insights. For more information, visit www.3x4genetics.com

    About Rupa Health

    Rupa Health is on a mission to bring root-cause medicine to every person on the planet. By simplifying the process of ordering, managing and interpreting diagnostic tests, Rupa helps practitioners identify and address the underlying factors that contribute to illness, empowering them to deliver personalized, data-driven, and holistic care. Rupa also brings the latest root cause medicine insights and offers modern patient experience. For more information, visit Rupahealth.com

    #Genomics, #DNAScience, #GeneticsResearch, #CRISPR, #GeneExpression, #GenomeEditing, #Epigenetics, #GeneticDiversity, #MolecularGenetics, #PopulationGenetics, #GeneticInheritance, #Bioinformatics, #GeneticMarkers, #GeneTherapy, #GeneticEngineering, #PrecisionMedicine, #GeneticTesting, #Pharmacogenomics, #MitochondrialDNA, #GeneticCounseling

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    October 30, 2024

    Genetic Landscape of Cancer Drug

    Base editing screens define the genetic landscape of cancer drug resistance mechanisms


    Drug resistance is a principal limitation to the long-term efficacy of cancer therapies. Cancer genome sequencing can retrospectively delineate the genetic basis of drug resistance, but this requires large numbers of post-treatment samples to nominate causal variants. Here we prospectively identify genetic mechanisms of resistance to ten oncology drugs from CRISPR base editing mutagenesis screens in four cancer cell lines using a guide RNA library predicted to install 32,476 variants in 11 cancer genes. We identify four functional classes of protein variants modulating drug sensitivity and use single-cell transcriptomics to reveal how these variants operate through distinct mechanisms, including eliciting a drug-addicted cell state. We identify variants that can be targeted with alternative inhibitors to overcome resistance and functionally validate an epidermal growth factor receptor (EGFR) variant that sensitizes lung cancer cells to EGFR inhibitors. Our variant-to-function map has implications for patient stratification, therapy combinations and drug scheduling in cancer treatment.

    Main

    Resistance to molecularly targeted anti-cancer treatments remains a major clinical challenge1. Drug resistance is frequently caused by DNA single nucleotide variants (SNVs) in the cancer genome2, leading to point mutations in the drug target or proteins within the same signaling pathway3. The study of drug resistance can inform drug mechanism of action, the design of second-generation inhibitors targeting drug-resistant proteins, the development of combination therapies and patient stratification for second-line therapies. Current approaches often depend on sequencing tumor biopsies from patients that relapse on treatment. These are challenging samples to acquire, meaning it can take years to accrue enough to infer variant function. These analyses are generally restricted to frequently observed variants and must be individually experimentally validated to establish a causal link to drug resistance. This is a slow process that does not allow for the direct comparison of different variant effects. These challenges limit the interpretation of cancer biopsy and circulating tumor DNA sequencing data. Rapid, prospective and systematic functional annotation of variants would accelerate the discovery of drug resistance mechanisms.

    CRISPR-based gene editing approaches such as base editing can be used to directly interpret the function of variants of unknown significance (VUS)4,5,6,7,8,9,10,11,12 and study genetic mechanisms of resistance to cytokines and inhibitors8,13,14. Cytidine and adenine base editors use a Cas9 nickase fused to a deaminase, facilitating the programmed installation of C>T and A>G SNVs in the genome at high efficiency in physiologically relevant cell types9,10,11,12,15,16,17,18. Here we use base editing at scale to investigate genetic mechanisms of acquired resistance to molecularly targeted cancer therapies, identifying VUS conferring drug resistance and drug sensitization in cancer cells. We classify cancer variants modulating drug sensitivity into four functional classes, thus providing a systematic framework for interpreting drug resistance mechanisms.

    Results

    Base editing screens map functional domains in cancer genes


    We investigated drug resistance to ten molecularly targeted cancer drugs that are currently approved by the United States Food and Drug Administration (FDA) or are under clinical investigation (Fig. 1a). We selected four cancer cell lines that are sensitive to these agents19 and harbor diverse oncogenic drivers20: H23 (lung; KRAS-G12C), PC9 (lung; EGFR amplification and exon19 deletion), HT-29 (colon; BRAF V600E) and MHH-ES-1 (Ewing sarcoma; EWS-FLI1 fusion). We mutagenized 11 cancer genes that encode common drug targets or genes within the same signaling pathway for interrogation with a guide RNA (gRNA) library (n = 22,816), tiling these genes and their 5′ and 3′ untranslated regions (UTRs). As controls, we included nontargeting gRNAs (NT gRNAs) (n = 57), intergenic-targeting gRNAs (n = 168) and gRNAs predicted to introduce splice variants21 in nonessential (n = 87) and essential (n = 316) genes22,23 (Supplementary Table 1). To maximize the saturation of targeted mutagenesis, the gRNA library was introduced into cancer cell lines expressing doxycycline-inducible cytidine base editor (CBE) or adenine base editor (ABE)8 with relaxed PAM requirements (Cas9–NGN)24. We analyzed the potential functional effects of thousands of gene variants on drug resistance in parallel by performing base editing screens with a proliferation read-out in the presence of targeted anti-cancer drugs from 46 independent pooled genetic screens (Fig. 1a and Supplementary Table 2). Base editing screen replicates were highly correlated (Extended Data Fig. 1), and control gRNAs targeting essential genes were depleted, indicating efficient base editing (Fig. 1b and Extended Data Fig. 2a).
    genetic landscape, cancer, drug response, genetic mutations, drug resistance, cancer therapies, tumor growth, personalized treatment, drug metabolism, next-generation sequencing, molecular profiling, actionable mutations, precision oncology, treatment efficacy, adverse effects, genetic variations, targeted therapies, therapeutic resistance, cancer genomics, molecular oncology,

    #CancerGenetics, #DrugResponse, #GeneticMutations, #CancerTherapy, #DrugResistance, #TumorGrowth, #PersonalizedTreatment, #DrugMetabolism, #NextGenSequencing, #MolecularProfiling, #ActionableMutations, #PrecisionOncology, #TreatmentEfficacy, #GeneticVariations, #TargetedTherapies, #TherapeuticResistance, #CancerGenomics, #MolecularOncology, #CancerResearch, #OncologyBreakthroughs

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      Scientists Identify Six Novel Genes

      Scientists identify six novel genes linked to cancer risk


      Scientists at deCODE genetics/Amgen, and their collaborators have discovered six novel genes with rare germline variants that associate with cancer risk. The findings are published today in Nature Genetics under the title "Gene-based burden tests of rare germline variants identify six cancer susceptibility genes."

      A subset of cancers arises in individuals who are born with rare sequence variants that significantly alter their cancer risk. The discovery of such variants, like those in the BRCA1- and BRCA2 genes, has led to improved early cancer detection and the development of targeted therapies, ultimately reducing the cancer burden and improving prognosis of those carrying these mutations.

      In this study, the scientists analyzed three large genetic datasets from individuals of European descent, including 130,991 cancer patients and 733,486 controls.

      Through a gene-based burden association analysis across 22 different cancer types, they found four novel genes associated with a risk of developing cancer; the pro-apoptotic BIK for prostate cancer, the autophagy involved ATG12 for colorectal cancer, TG for thyroid cancer, and CMTR2 for both lung cancer and cutaneous melanoma.

      The relative increase in cancer risk conferred by these variants was substantial (90%–295%), but it should be noted that the design of the study does not allow accurate assessment of absolute lifetime cancer risk.

      Additionally, the researchers found the first genes with rare variants that are associated with a decreased risk of cancer. Specifically, loss of AURKB was found to protect against any cancer type, and loss of PPP1R15A was associated with a 53% lower risk of breast cancer. This suggests that inhibition of PPP1R15A may be a therapeutic option for breast cancer.

      The study revealed new insight into the biological mechanisms involved in cancer predisposition that will hopefully lead to better screening and treatment strategies.

      cancer genetics, gene discovery, cancer risk, novel genes, cancer susceptibility, molecular mechanisms, genetic mutations, early detection, personalized treatment, cancer research, risk assessment, precision medicine, targeted therapy, cancer prevention, oncogenes, tumor suppressor genes, genomic sequencing, cancer biomarkers, genetic screening, healthcare innovation,

      #CancerGenetics, #GeneDiscovery, #CancerRisk, #NovelGenes, #GeneticResearch, #CancerSusceptibility, #MolecularMechanisms, #GeneticMutations, #EarlyDetection, #PersonalizedMedicine, #CancerResearch, #RiskAssessment, #PrecisionMedicine, #TargetedTherapy, #CancerPrevention, #Oncogenes, #TumorSuppressor, #GenomicSequencing, #CancerBiomarkers, #GeneticScreening

      International Conference on Genetics and Genomics of Diseases 




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      October 28, 2024

      Anti-Tumor Effects

      A PD-L1 tropism-expanded oncolytic adenovirus enhanced gene delivery efficiency and anti-tumor effects

      Recombinant adenovirus serotype 5 (Ad5)-mediated virotherapy is a maturing technique in cancer treatment. However, the utility of adenovirus (Ad) has been limited by low expression of coxsackievirus and adenovirus receptor (CAR) in cancer cells resulting in poor infectivity of Ads. To overcome the problem, we aimed to develop a novel tropism-modified oncolytic adenovirus, ZD55-F-HI-sPD-1-EGFP, which contains the epitope of PD-1 (70-77aa) at the HI-loop of Ad fiber. Trimerization of Fiber-sPD-1 was confirmed by immunoblot analysis. ZD55-F-HI-sPD-1-EGFP shows a remarkable improvement in viral infection rate and gene transduction efficiency in the PD-L1-positive cancer cells. Competition assays with a PD-L1 protein reveals that cell internalization of ZD55-F-HI-sPD-1-EGFP is mediated by both CAR and PD-L1 at a high dose. The progeny virus production capacity showed that sPD-1 incorporated fiber-modified oncolytic Ad replication was not affected. Furthermore, treating with ZD55-F-HI-sPD-1-EGFP significantly increased viral infection rate and enhanced anti-tumor effect in vivo. This study demonstrates that the strategy to expand tropism of oncolytic Ad may significantly improve therapeutic profile for cancer treatment.

      Oncolytic virotherapy is considered as one of the most promising cancer therapies, because of their ability to selectively replicate and lyse tumor cells, induce inflammatory response, release tumor-associated antigens to activate host’s adaptive anti-tumor immunity, and promote lymphocyte infiltration in tumors [1], [2], [3], [4], [5]. Adenovirus serotype 5 (Ad5) based oncolytic adenovirus are widely applied to pre-clinical and clinical trials [6], [7]. Ad5 infection of cells is initiated by the formation of a complex between the fiber protein knob domain of the virus and the host cell surface coxsackievirus and adenovirus receptor (CAR) [8], [9]. However, CAR is expressed at low levels in many types of cancer cells [10], [11], [12], [13], which limits the infectivity and oncolytic efficiency of Ad5-mediated oncolytic adenovirus [14], [15]. Therefore, the methods to increase infectivity of Ad5 are required to maximize the therapeutic efficacy of oncolytic Ad in cancers with CAR-deficient.
      To broaden the native tropism of oncolytic Ad, several genetic modifications of Ads have been studied, which are mainly focused on fiber proteins. Considering that the tropism of infection often differed between adenovirus subgroups [16], fiber knob serotype switching is one of the main approaches to broaden the tropism of Ad5-based vectors. Numerous Ad5-based oncolytic vectors with chimeric fibers have been developed, such as replacement of Ad5 knob to Ad37 enhanced viral infectivity against glioma cell [17], and replacement of Ad5 knob to Ad35 knob enhanced CD46-dependent Ad gene transfer efficiency [18]. Chimeric fibers of Ad5/3 [19], [20] and Ad5/35 [21], [22] also have been developed as cancer therapies. In addition, insertion of a peptide to the C-terminus or HI-loop of the fiber protein is another main approach to expand the tropism of Ad5-based vectors. Incorporating the vesicular stomatitis virus glycoprotein (VSV-G) epitope onto the C terminus of the fiber knob facilitated the specificity binding of the virus to phosphatidylserine (PS) moieties on the cellular plasma membrane [23]. Addition a cationic polylysine sequence (pK7) to the fiber knob facilitated viral binding to negatively charged cell surface molecules, such as heparin sulfates and increased Ad infection to endothelial cells and fibroblasts [24]. Similarly, insertion a peptide of arginine-glycineaspartate (RGD) integrin recognition site to the C-terminus or HI-loop of the fiber protein signally increased Ad infection in wide range of cells such as fibroblasts, endothelial cells, and smooth muscle cells [25]. However, the current fiber protein modification aimed at increasing the Ad infectivity cannot satisfy all tumor cell types due to the diversity of tumors. Therefore, more fiber protein modification studies are needed to expand the tropism of adenovirus to invade different tumor cells. Structure of the complex of human PD-1 and its ligand PD-L1 showed that the 70–77 amino acid peptide (70 to 77, MSPSNQTD) of PD-1 plays a role in interacting with PD-L1 [26]. Therefore, we hypothesized that insertion short PD-1 (sPD-1, 70-77aa) to HI-loop of the fiber knob can expand the tropism of Ad to PD-L1 positive tumor cells.

      Here, we constructed a tropism expanded oncolytic adenovirus (ZD55-F-HI-sPD-1-EGFP), which incorporated a short peptide of PD-1 (sPD-1, 70-77aa) in HI-loop of fiber knob and contained an exogenous reporter gene, EGFP. We verified that fiber trimer formation and viral replication ability of ZD55-F-HI-sPD-1-EGFP were not affected compared to the control virus ZD55-EGFP with wild-type fiber. ZD55-F-HI-sPD-1-EGFP increased viral infectivity, gene transduction efficiency and antitumoral efficacy, in comparison to control oncolytic adenovirus, ZD55-EGFP, in PD-L1-positive cancer cells. This is the first report to demonstrate sPD-1 epitope incorporated oncolytic Ad has expanded tropism that can improve viral infection and gene transduction efficiency in PD-L1-positive cancer cells and strengthened its therapeutic applications.

      #PDL1, #OncolyticAdenovirus, #GeneDelivery, #AntiTumor, #CancerImmunotherapy, #PrecisionOncology, #ImmuneResponse, #AdenovirusEngineering, #TumorTargeting, #GeneTherapy, #CancerTreatment, #ViralTropism, #OncolyticVirus, #ImmuneCheckpoint, #PDL1Expression, #TumorLysis, #TargetedTherapy, #AdenoviralVectors, #TherapeuticGenes, #Oncology

      International Conference on Genetics and Genomics of Diseases 




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      Genetics and Genomic Breeding

      Grapevine pangenome facilitates trait genetics and genomic breeding Grapevine breeding is hindered by a limited understanding of the genetic...