CRISPR-Cas9 globin editing can induce megabase-scale copy-neutral losses of heterozygosity in hematopoietic cells.
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ABSTRACT:
SUBMITTER: Boutin J
PROVIDER: S-EPMC8363739 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundA KIT gain of function mutation is present in 70% of gastrointestinal stromal tumors (GISTs) and the wild-type (WT) allele is deleted in 5 to 15% of these cases. The WT KIT is probably deleted during GIST progression. We aimed to identify the mechanism of WT KIT loss and to determine whether other genes are involved or affected.MethodsWhole-genome SNP array analyses were performed in 22 GISTs with KIT exon 11 mutations, including 11 with WT loss, to investigate the mechanisms of WT allele deletion. CGH arrays and FISH were performed in some cases. Common genetic events were identified by SNP data analysis. The 9p21.3 locus was studied by multiplex quantification of genomic DNA.ResultsChromosome instability involving the whole chromosome/chromosome arm (whole C/CA) was detected in 21/22 cases. The GISTs segregated in two groups based on their chromosome number: polyGISTs had numerous whole C/CA gains (mean 23, range [9 to 43]/3.11 [1 to 5]), whereas biGISTs had fewer aberrations. Whole C/CA losses were also frequent and found in both groups. There were numerous copy-neutral losses of heterozygosity (cnLOH) of whole C/CA in both polyGIST (7/9) and biGIST (9/13) groups. cnLOH were frequent on 4q, 11p, 11q, 1p, 2q, 3p and 10, and never involved 12p, 12q, 20p, 20q or 19q. Other genetic alterations included segmental chromosome abnormalities, complete bi-allelic deletions (homozygous deletions) and, more rarely, amplifications. Nine of 11 GISTs with homozygous KIT exon 11 mutations had cnLOH of chromosome 4.ConclusionThe cnLOH of whole C/CA is a frequent genetic alteration in GISTs and is closely associated with homozygous mutations of KIT and WT allele deletion.
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Project description:CRISPR-Cas9 is a promising technology for genome editing. Here we use Cas9 nuclease-induced double-strand break DNA (DSB) at the UROS locus to model and correct congenital erythropoietic porphyria. We demonstrate that homology-directed repair is rare compared with NHEJ pathway leading to on-target indels and causing unwanted dysfunctional protein. Moreover, we describe unexpected chromosomal truncations resulting from only one Cas9 nuclease-induced DSB in cell lines and primary cells by a p53-dependent mechanism. Altogether, these side effects may limit the promising perspectives of the CRISPR-Cas9 nuclease system for disease modeling and gene therapy. We show that the single nickase approach could be safer since it prevents on- and off-target indels and chromosomal truncations. These results demonstrate that the single nickase and not the nuclease approach is preferable, not only for modeling disease but also and more importantly for the safe management of future CRISPR-Cas9-mediated gene therapies.
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Project description:β-thalassemias (β-thal) are a group of blood disorders caused by mutations in the β-globin gene (HBB) cluster. β-globin associates with α-globin to form adult hemoglobin (HbA, α2β2), the main oxygen-carrier in erythrocytes. When β-globin chains are absent or limiting, free α-globins precipitate and damage cell membranes, causing hemolysis and ineffective erythropoiesis. Clinical data show that severity of β-thal correlates with the number of inherited α-globin genes (HBA1 and HBA2), with α-globin gene deletions having a beneficial effect for patients. Here, we describe a novel strategy to treat β-thal based on genome editing of the α-globin locus in human hematopoietic stem/progenitor cells (HSPCs). Using CRISPR/Cas9, we combined 2 therapeutic approaches: (1) α-globin downregulation, by deleting the HBA2 gene to recreate an α-thalassemia trait, and (2) β-globin expression, by targeted integration of a β-globin transgene downstream the HBA2 promoter. First, we optimized the CRISPR/Cas9 strategy and corrected the pathological phenotype in a cellular model of β-thalassemia (human erythroid progenitor cell [HUDEP-2] β0). Then, we edited healthy donor HSPCs and demonstrated that they maintained long-term repopulation capacity and multipotency in xenotransplanted mice. To assess the clinical potential of this approach, we next edited β-thal HSPCs and achieved correction of α/β globin imbalance in HSPC-derived erythroblasts. As a safer option for clinical translation, we performed editing in HSPCs using Cas9 nickase showing precise editing with no InDels. Overall, we described an innovative CRISPR/Cas9 approach to improve α/β globin imbalance in thalassemic HSPCs, paving the way for novel therapeutic strategies for β-thal.
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Project description:Despite improvements over the past 20 years, African Americans continue to have the highest incidence and mortality rates of colorectal cancer (CRC) in the United States. While previous studies have found that copy number variations (CNVs) occur at similar frequency in African American and White CRCs, copy-neutral loss of heterozygosity (cnLOH) has not been investigated. In the present study, we used publicly available data from The Cancer Genome Atlas (TCGA) as well as data from an African American CRC cohort, the Chicago Colorectal Cancer Consortium (CCCC), to compare frequencies of CNVs and cnLOH events in CRCs in the two racial groups. Using genotype microarray data, we analyzed large-scale CNV and cnLOH events from 166 microsatellite stable CRCs-31 and 39 African American CRCs from TCGA and the CCCC, respectively, and 96 White CRCs from TCGA. As reported previously, the frequencies of CNVs were similar between African American and White CRCs; however, there was a significantly lower frequency of cnLOH events in African American CRCs compared to White CRCs, even after adjusting for demographic and clinical covariates. Although larger differences for chromosome 18 were observed, a lower frequency of cnLOH events in African American CRCs was observed for nearly all chromosomes. These results suggest that mechanistic differences, including differences in the frequency of cnLOH, could contribute to clinicopathological disparities between African Americans and Whites. Additionally, we observed a previously uncharacterized phenomenon we refer to as small interstitial cnLOH, in which segments of chromosomes from 1 to 5 Mb long were affected by cnLOH.
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Project description:Loss of heterozygosity (LOH) is a common type of genomic alterations in ovarian cancer. Analyzing 74,415 copy neutral LOH events in 513 serous ovarian adenocarcinomas samples from the Cancer Genome Atlas, we report that the frequency of LOH events increases with age. Similar trend is observed for LOH involving chromosome 17, which is frequently implicated in ovarian cancer. The results are consistent when we analyze data from the Boston high-grade serous cancer cohort. We further show that germ line and somatic mutations in BRCA1 (in chromosome 17) and BRCA2 (in chromosome 13) loci are not necessary to establish the pattern. We also report significant age-related changes in expression patterns for several genes in the homologous recombination (HR) pathway, such as BRCA1, RAD50, RAD52, XRCC2, XRCC3, and MRE11A in these patient samples. Furthermore, we develop a metric for pathway-level imbalance, and show that increased imbalance in the HR pathway, i.e., increase in expression of some HR genes and decrease in expression of others, is common and correlates significantly with the frequency of LOH events in the patient samples. Taken together, it is highly likely that aging and deregulation of HR pathway contribute to the increased incidence of copy-neutral LOH in ovarian cancer patients.
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Project description:Somatic mosaicism denotes the presence of genetically distinct populations of somatic cells in one individual who has developed from a single fertilised oocyte. Mosaicism may result from a mutation that occurs during postzygotic development and is propagated to only a subset of the adult cells. Our aim was to investigate both somatic mosaicism for copy-neutral loss of heterozygosity (cn-LOH) events and DNA copy number variations (CNVs) in fully differentiated tissues.We studied panels of tissue samples (11-12 tissues per individual) from four autopsy subjects using high-resolution Illumina HumanOmniExpress-12 BeadChips to reveal the presence of possible intra-individual tissue-specific cn-LOH and CNV patterns. We detected five mosaic cn-LOH regions >5 Mb in some tissue samples in three out of four individuals. We also detected three CNVs that affected only a portion of the tissues studied in one out of four individuals. These three somatic CNVs range from 123 to 796 kb and are also found in the general population. An attempt was made to explain the succession of genomic events that led to the observed somatic genetic mosaicism under the assumption that the specific mosaic patterns of CNV and cn-LOH changes reflect their formation during the postzygotic embryonic development of germinal layers and organ systems.Our results give further support to the idea that somatic mosaicism for CNVs, and also cn-LOHs, is a common phenomenon in phenotypically normal humans. Thus, the examination of only a single tissue might not provide enough information to diagnose potentially deleterious CNVs within an individual. During routine CNV and cn-LOH analysis, DNA derived from a buccal swab can be used in addition to blood DNA to get information about the CNV/cn-LOH content in tissues of both mesodermal and ectodermal origin. Currently, the real frequency and possible phenotypic consequences of both CNVs and cn-LOHs that display somatic mosaicism remain largely unknown. To answer these questions, future studies should involve larger cohorts of individuals and a range of tissues.
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Project description:Genetic manipulation is often used to study gene function. However, unplanned genome changes (including single nucleotide polymorphisms [SNPs], aneuploidy, and loss of heterozygosity [LOH]) can affect the phenotypic traits of the engineered strains. Here, we compared the effect of classical deletion methods (replacing target alleles with selectable markers by homologous recombination) with CRISPR-Cas9 editing in the diploid human-pathogenic yeast Candida parapsilosis. We sequenced the genomes of 9 isolates that were modified using classic recombination methods and 12 that were edited using CRISPR-Cas9. As a control, the genomes of eight isolates that were transformed with a Cas9-expressing plasmid in the absence of a guide RNA were also sequenced. Following gene manipulation using classic homologous recombination, only one strain exhibited extensive LOH near the targeted gene (8.9 kb), whereas another contained multiple LOH events not associated with the intended modification. In contrast, large regions of LOH (up to >1,100 kb) were observed in most CRISPR-Cas9-edited strains. LOH most commonly occurred adjacent to the Cas9 cut site and extended to the telomere in four isolates. In two isolates, we observed LOH on chromosomes that were not targeted by CRISPR-Cas9. Among the CRISPR-edited isolates, two exhibited cysteine and methionine auxotrophy caused by LOH at a heterozygous site in MET10, approximately 11 and 157 kb downstream from the Cas9 target site, respectively. C. parapsilosis isolates have relatively low levels of heterozygosity. However, our results show that mutation complementation to confirm observed phenotypes is required when using CRISPR-Cas9. IMPORTANCE CRISPR-Cas9 has greatly streamlined gene editing and is now the gold standard and first choice for genetic engineering. However, we show that in diploid species, extra care should be taken in confirming the cause of any phenotypic changes observed. We show that the Cas9-induced double-strand break is often associated with loss of heterozygosity in the asexual diploid human fungal pathogen Candida parapsilosis. This can result in deleterious heterozygous variants (e.g., stop gain in one allele) becoming homozygous, resulting in unplanned phenotypic changes. Our results stress the importance of mutation complementation even when using CRISPR-Cas9.
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Project description:Anaplastic oligodendrogliomas (AOD) are rare glial tumors in adults with relative homogeneous clinical, radiological and histological features at the time of diagnosis but dramatically various clinical courses. Studies have identified several molecular abnormalities with clinical or biological relevance to AOD (e.g. t(1;19)(q10;p10), IDH1, IDH2, CIC and FUBP1 mutations).To better characterize the clinical and biological behavior of this tumor type, the creation of a national multicentric network, named "Prise en charge des OLigodendrogliomes Anaplasiques (POLA)," has been supported by the Institut National du Cancer (InCA). Newly diagnosed and centrally validated AOD patients and their related biological material (tumor and blood samples) were prospectively included in the POLA clinical database and tissue bank, respectively.At the molecular level, we have conducted a high-resolution single nucleotide polymorphism array analysis, which included 83 patients. Despite a careful central pathological review, AOD have been found to exhibit heterogeneous genomic features. A total of 82% of the tumors exhibited a 1p/19q-co-deletion, while 18% harbor a distinct chromosome pattern. Novel focal abnormalities, including homozygously deleted, amplified and disrupted regions, have been identified. Recurring copy neutral losses of heterozygosity (CNLOH) inducing the modulation of gene expression have also been discovered. CNLOH in the CDKN2A locus was associated with protein silencing in 1/3 of the cases. In addition, FUBP1 homozygous deletion was detected in one case suggesting a putative tumor suppressor role of FUBP1 in AOD.Our study showed that the genomic and pathological analyses of AOD are synergistic in detecting relevant clinical and biological subgroups of AOD.
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Project description:Myelodysplastic syndromes (MDS) are a heterogeneous group of diseases with varying genetic aberrations. Half of MDS patients have normal karyotype, obscuring the underlying condition indicating a need for new markers for improved diagnostics and prognosis. We performed a retrospective review of sequential MDS patients who underwent chromosomal genetic array testing (CGAT) between November 2008 and March 2014. Total Genomic Aberration (TGA) scores, with and without copy-neutral loss of heterozygosity (cnLOH), were compared to pathology and clinical data. Of 68 MDS participants, 50 patients (73%) had abnormal CGAT results. 32% showedcnLOH, 41% had no cnLOH but displayed copy number aberration (CNAs). Of 26 patients with normal cytogenetics, 46% had clonal abnormalities by CGAT. Abnormal CGAT results were associated with lower overall survival (P=0.04). Overall survival in patients with TGA above the median (68.6 Mb) was significantly inferior to those below the median (HR=2.9, 95% CI=1.3-6.8, P=0.01). Furthermore, there was an observed association between increased TGA and increased dysplastic lineages (Ptrend=0.003). CGAT studies provide important findings that extend beyond current standard testing. Clinical utility of CGAT includes improved diagnostic yield, correlation of extent of TGA and increased dysplastic features, and survival.
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