| 1. |
- Larsson, Chatarina, et al.
(författare)
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DIP2C regulates expression of the tumor suppressor gene CDKN2A
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The disco-interacting protein 2 homolog C (DIP2C) gene is an uncharacterized candidatebreast and lung cancer gene. The gene contains a DMAP1 binding domain, pointing topotential involvement in DNMT1-dependent methylation. To study the role of DIP2C intumor development, we engineered human DIP2C knockout cell systems by rAAV-mediatedgene targeting. Homo- and heterozygous RKO DIP2C knockout cells displayed enlarged cellsand growth retardation. This phenotype was most pronounced in DIP2C-/- knockouts, andthese cells also displayed a significant decrease in DIP2C mRNA levels. RNA sequencingrevealed 780 genes affected by the loss of DIP2C, including the cellular senescence markerP16INK4a. Functional annotation of the regulated genes shows enrichment of genes involvedwith cell death processes, cell structure and motility. Furthermore, KEGG pathway analysisshows association of 19 genes with pathways in cancer. In conclusion, the phenotypic dataand expression changes induced by loss of DIP2C indicate that the gene function may beimportant for several biological processes implicated in cancer, and that loss of gene functionmay be a trigger of cellular senescence.
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| 2. |
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| 3. |
- Chatzakos, Vicky, 1977-, et al.
(författare)
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N-Acyl Taurines are Anti-Proliferative in Prostate Cancer Cells
- 2012
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Ingår i: Lipids. - : Wiley. - 0024-4201 .- 1558-9307. ; 47:4, s. 355-361
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Tidskriftsartikel (refereegranskat)abstract
- Endocannabinoids have been implicated in cancer development and cause heterogenous effects in tumor cells, by inducing apoptosis, reducing migration, causing anti-angiogenic activity and alterations in the cell cycle resulting in growth arrest. Recently, several novel amides of fatty acids that are structurally related to endocannabinoids have been isolated from mammalian sources, although the functions of these fatty amides are not well studied. One group of these novel fatty acid amides are the N-acyl taurines (fatty acids conjugated to the amino acid taurine). This study examined if N-acyl taurines, specifically N-arachidonoyl taurine and N-oleoyl taurine could function in a similar way to endocannabinoids and result in cell cycle alterations or growth arrest in the human prostate adenocarcinoma cell line PC-3. PC-3 cells were treated with various concentrations of N-arachidonoyl taurine and N-oleoyl taurine and cell proliferation and viability was measured using resazurin and colony formation assays. Effects of N-acyl taurines on the cell cycle was measured using FACS analysis. Treatment with N-arachidonoyl taurine and N-oleoyl taurine resulted in a significant reduction in proliferation of PC-3 cells, even at concentrations as low as 1 mu M. Treatment with N-oleoyl taurine resulted in an increased number of cells in the subG1 population, suggesting apoptosis, and a lower number of cells in S-phase of the cell cycle. In summary, our results show that novel biologically active lipids, the N-acyl taurines, result in reduced proliferation in PC-3 cells.
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| 4. |
- Djureinovic, Tatjana
(författare)
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Investigation of genetic factors involved in colorectal cancer predisposition
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Colorectal cancer (CRC) is one of the leading causes of cancer-related death in the western world. Several hereditary CRC predisposing syndromes have been described and their genetic component has been elucidated. Taken together, these syndromes account for at most 5% of all CRC cases. Twin studies have, however, suggested a much stronger contribution of genetic factors suggesting that a number of CRC predisposing genes remain to be identified. The genome-wide linkage analysis in 18 non-FAP/non-HNPCC colorectal cancer families revealed regions of interest on three chromosomes, when the analysis was performed under the assumption of locus heterogeneity. The region on chromosome 22q was suggested in the parametric analysis, while the results of both parametric and nonparametric analysis provided support for the regions on chromosomes 11q and 14q. After finemapping of the regions on chromosomes 11q and 14q both the HLOD score and the NPL scores were reduced but still within the range of suggestive linkage. Families exhibiting linkage to chromosomes 11 and 14 were identified and overlapping regions were determined (1 1q13.2-13.4, 1 1q22.1- 23.1 and 14q23.1-24.1). (Paper I) The SMARCA3 gene has been shown to be a common target for methylation in colon and gastric cancer. Germline mutation screening of the SMARCA3 gene was performed in order to evaluate the role of this gene among Swedish colorectal cancer patients, some of whom also had a family history of gastric cancer. The lack of pathogenic germline mutations suggests that the gene has very little role, if any, in the predisposition to colorectal and gastric cancer. Several identified variants in the SMARCA3 gene could act as modifying or low-risk alleles, however additional studies are needed to determine their role. (Paper II) The CHEK2 1100delC, novel low-risk breast cancer predisposing allele, has been found at particularly high frequency in families with both colorectal and breast cancer. To investigate the possible role of this variant in CRC predisposition the variant frequency was determined in CRC cases and controls. No over-representation of the variant was detected in cases. However, due to the low frequency of the variant in the Swedish population, a very low penetrance effect of CHEK2 1100delC could not be excluded. (Paper III) Bi-allelic germline mutations in the MUTYH gene are known to predispose to recessively inherited MAP syndrome, characterized by the occurrence of an increased number of colonic polyps. We found no evidence for the contribution of MUTYH in familial CRC with a low polyp number. The two most common MUTYH mutations, Y165C and G382D, have been identified in the Swedish population and evidence for a slightly increased CRC risk among heterozygote carriers of these mutations was seen. In addition, three novel variants affecting the same amino acid position, R423Q, R423P and R423R, have been detected among sporadic CRC cases, however their significance remains to be determined. (Paper IV)
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| 5. |
- Djureinovic, Tatjana, et al.
(författare)
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The CHEK2 1100delC variant in Swedish colorectal cancer
- 2006
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Ingår i: Anticancer Research. - 0250-7005 .- 1791-7530. ; 26:6C, s. 4885-4888
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The cell cycle checkpoint kinase 2 (CHEK2) 1100delC variant has recently been identified at high frequency in families with both breast and colorectal cancer, suggesting the possible role of this variant in colorectal cancer predisposition. PATIENTS AND METHODS: To evaluate the role of CHEK2 ll00delC among Swedish colorectal cancer patients, the variant frequency was determined in 174 selected familial cases, 644 unselected cases and 760 controls, as well as in l8 families used in the genome-wide linkage analysis, where weak linkage was seen for the region harboring the CHEK2 gene. RESULTS: CHEK2 l100delC was found in 1.15% of familial and in 0.93% of unselected cases, compared to 0.66% of controls, showing no significant difference between groups. One out of 45 familial cases with a family history of breast cancer was shown to be a carrier. The variant was not identified in the 18 families included in the linkage analysis. CONCLUSION: The CHEK2 1100delC was not significantly increased in Swedish colorectal cancer patients, however, in order to determine the role of the variant in colorectal cancer families with the history of breast cancer a larger sample size is needed.
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| 6. |
- Elvers, Ingegerd, et al.
(författare)
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CHK1 activity is required for continuous replication fork elongation but not stabilization of post-replicative gaps after UV irradiation
- 2012
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 40:17, s. 8440-8448
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Tidskriftsartikel (refereegranskat)abstract
- Ultraviolet (UV)-induced DNA damage causes an efficient block of elongating replication forks. The checkpoint kinase, CHK1 has been shown to stabilize replication forks following hydroxyurea treatment. Therefore, we wanted to test if the increased UV sensitivity caused by the unspecific kinase inhibitor caffeine-inhibiting ATM and ATR amongst other kinases-is explained by inability to activate the CHK1 kinase to stabilize replicative structures. For this, we used cells deficient in polymerase eta (Pol eta), a translesion synthesis polymerase capable of properly bypassing the UV-induced cis-syn TT pyrimidine dimer, which blocks replication. These cells accumulate gaps behind progressing replication forks after UV exposure. We demonstrate that both caffeine and CHK1 inhibition, equally retards continuous replication fork elongation after UV treatment. Interestingly, we found more pronounced UV-sensitization by caffeine than with the CHK1 inhibitor in clonogenic survival experiments. Furthermore, we demonstrate an increased collapse of replicative structures after caffeine treatment, but not after CHK1 inhibition, in UV-irradiated cells. This demonstrates that CHK1 activity is not required for stabilization of gaps induced during replication of UV-damaged DNA. These data suggest that elongation and stabilization of replicative structures at UV-induced DNA damage are distinct mechanisms, and that CHK1 is only involved in replication elongation.
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| 7. |
- Elvers, Ingegerd, et al.
(författare)
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CHK1 activity is required for replication fork elongation but not stabilisation after UV irradiation
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Tidskriftsartikel (refereegranskat)abstract
- UV-induced DNA damage cause an efficient block for elongating replication forks. Since CHK1 has been shown to stabilise replication forks following hydroxyurea treatment, we wanted to test if the increased killing with the unspecific kinase inhibitor caffeine, inhibiting ATM and ATR amongst other kinases, is explained by inability to activate the CHK1 kinase to stabilise UV-stalled replication forks. For this, we used cells deficient in Polη, a translesion synthesis polymerase capable of properly bypassing the UV-induced cis-syn TT pyrimidine dimer, which blocks replication. These cells, derived from the variant type of xeroderma pigmentosum, are sensitised to UV irradiation by caffeine treatment. We demonstrate that both caffeine and CHK1 inhibition, using CEP-3891, equally retards replication fork elongation after UV treatment in Polη deficient cells. Interestingly, we found more pronounced UV-sensitisation by caffeine than with the CHK1 inhibitor in clonogenic survival experiments. Furthermore, we demonstrate an increased collapse of UV-stalled forks after caffeine treatment, but not after CHK1 inhibition, demonstrating that CHK1 activity is not required for stabilisation of UV-stalled replication forks. These data suggest that stabilisation and elongation at UV-stalled forks are distinct mechanisms, and that CHK1 is only involved in fork elongation.
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| 8. |
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| 9. |
- Gad, Helge, et al.
(författare)
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MTH1 inhibition eradicates cancer by preventing sanitation of the dNTP pool
- 2014
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Ingår i: Nature. - : Nature Publishing Group. - 0028-0836 .- 1476-4687. ; 508:7495, s. 215-221
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Tidskriftsartikel (refereegranskat)abstract
- Cancers have dysfunctional redox regulation resulting in reactive oxygen species production, damaging both DNA and free dNTPs. The MTH1 protein sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Although MTH1 is non-essential in normal cells, we show that cancer cells require MTH1 activity to avoid incorporation of oxidized dNTPs, resulting in DNA damage and cell death. We validate MTH1 as an anticancer target in vivo and describe small molecules TH287 and TH588 as first-in-class nudix hydrolase family inhibitors that potently and selectively engage and inhibit the MTH1 protein in cells. Protein co-crystal structures demonstrate that the inhibitors bindin the active site of MTH1. The inhibitors cause incorporation of oxidized dNTPs in cancer cells, leading to DNA damage, cytotoxicity and therapeutic responses in patient-derived mouse xenografts. This study exemplifies the non-oncogene addiction concept for anticancer treatment and validates MTH1 as being cancer phenotypic lethal.
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| 10. |
- Gottipati, Ponnari, et al.
(författare)
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Poly(ADP-Ribose) Polymerase Is Hyperactivated in Homologous Recombination-Defective Cells
- 2010
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Ingår i: Cancer Research. - 0008-5472 .- 1538-7445. ; 70:13, s. 5389-5398
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Tidskriftsartikel (refereegranskat)abstract
- Poly(ADP-ribose) (PAR) polymerase 1 (PARP1) is activated by DNA single-strand breaks (SSB) or at stalled replication forks to facilitate DNA repair. Inhibitors of PARP efficiently kill breast, ovarian, or prostate tumors in patients carrying hereditary mutations in the homologous recombination (HR) genes BRCA1 or BRCA2 through synthetic lethality. Here, we surprisingly show that PARP1 is hyperactivated in replicating BRCA2-defective cells. PARP1 hyperactivation is explained by the defect in HR as shRNA depletion of RAD54, RAD52, BLM, WRN, and XRCC3 proteins, which we here show are all essential for efficient HR and also caused PARP hyperactivation and correlated with an increased sensitivity to PARP inhibitors. BRCA2-defective cells were not found to have increased levels of SSBs, and PAR polymers formed in HR-defective cells do not colocalize to replication protein A or gamma H2AX, excluding the possibility that PARP hyperactivity is due to increased SSB repair or PARP induced at damaged replication forks. Resistance to PARP inhibitors can occur through genetic reversion in the BRCA2 gene. Here, we report that PARP inhibitor-resistant BRCA2-mutant cells revert back to normal levels of PARP activity. We speculate that the reason for the sensitivity of HR-defective cells to PARP inhibitors is related to the hyperactivated PARP1 in these cells. Furthermore, the presence of PAR polymers can be used to identify HR-defective cells that are sensitive to PARP inhibitors, which may be potential biomarkers. Cancer Res; 70(13); 5389-98. (C) 2010 AACR.
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| 11. |
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| 12. |
- Gubanova, Evgenia, et al.
(författare)
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SMG-1 suppresses CDK2 and tumor growth by regulating both the p53 and Cdc25A signaling pathways
- 2013
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Ingår i: Cell Cycle. - : Informa UK Limited. - 1538-4101 .- 1551-4005. ; 12:24, s. 3770-3780
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Tidskriftsartikel (refereegranskat)abstract
- The DNA damage response is coordinated by phosphatidylinositol 3-kinase-related kinases, ATM, ATR, and DNA-PK. SMG-1 is the least studied stress-responsive member of this family. Here, we show that SMG-1 regulates the G 1/S checkpoint through both a p53-dependent, and a p53-independent pathway. We identify Cdc25A as a new SMG-1 substrate, and show that cells depleted of SMG-1 exhibit prolonged Cdc25A stability, failing to inactivate CDK2 in response to radiation. Given an increased tumor growth following depletion of SMG-1, our data demonstrate a novel role for SMG-1 in regulating Cdc25A and suppressing oncogenic CDK2 driven proliferation, confirming SMG-1 as a tumor suppressor.
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| 13. |
- Jiao, Xiang, et al.
(författare)
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Gene rearrangements in hormone receptor negative breast cancers revealed by mate pair sequencing
- 2013
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Ingår i: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 14
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Tidskriftsartikel (refereegranskat)abstract
- Background: Chromosomal rearrangements in the form of deletions, insertions, inversions and translocations are frequently observed in breast cancer genomes, and a subset of these rearrangements may play a crucial role in tumorigenesis. To identify novel somatic chromosomal rearrangements, we determined the genome structures of 15 hormone-receptor negative breast tumors by long-insert mate pair massively parallel sequencing. Results: We identified and validated 40 somatic structural alterations, including the recurring fusion between genes DDX10 and SKA3 and translocations involving the EPHA5 gene. Other rearrangements were found to affect genes in pathways involved in epigenetic regulation, mitosis and signal transduction, underscoring their potential role in breast tumorigenesis. RNA interference-mediated suppression of five candidate genes (DDX10, SKA3, EPHA5, CLTC and TNIK) led to inhibition of breast cancer cell growth. Moreover, downregulation of DDX10 in breast cancer cells lead to an increased frequency of apoptotic nuclear morphology. Conclusions: Using whole genome mate pair sequencing and RNA interference assays, we have discovered a number of novel gene rearrangements in breast cancer genomes and identified DDX10, SKA3, EPHA5, CLTC and TNIK as potential cancer genes with impact on the growth and proliferation of breast cancer cells.
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| 14. |
- Larsson, Chatarina, 1979-, et al.
(författare)
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Loss of DIP2C in RKO cells stimulates changes in DNA methylation and epithelial-mesenchymal transition
- 2017
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Ingår i: BMC Cancer. - : BIOMED CENTRAL LTD. - 1471-2407. ; 17
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Tidskriftsartikel (refereegranskat)abstract
- Background: The disco-interacting protein 2 homolog C (DIP2C) gene is an uncharacterized gene found mutated in a subset of breast and lung cancers. To understand the role of DIP2C in tumour development we studied the gene in human cancer cells.Methods: We engineered human DIP2C knockout cells by genome editing in cancer cells. The growth properties of the engineered cells were characterised and transcriptome and methylation analyses were carried out to identify pathways deregulated by inactivation of DIP2C. Effects on cell death pathways and epithelial-mesenchymal transition traits were studied based on the results from expression profiling.Results: Knockout of DIP2C in RKO cells resulted in cell enlargement and growth retardation. Expression profiling revealed 780 genes for which the expression level was affected by the loss of DIP2C, including the tumour-suppressor encoding CDKN2A gene, the epithelial-mesenchymal transition (EMT) regulator-encoding ZEB1, and CD44 and CD24 that encode breast cancer stem cell markers. Analysis of DNA methylation showed more than 30,000 sites affected by differential methylation, the majority of which were hypomethylated following loss of DIP2C. Changes in DNA methylation at promoter regions were strongly correlated to changes in gene expression, and genes involved with EMT and cell death were enriched among the differentially regulated genes. The DIP2C knockout cells had higher wound closing capacity and showed an increase in the proportion of cells positive for cellular senescence markers.Conclusions: Loss of DIP2C triggers substantial DNA methylation and gene expression changes, cellular senescence and epithelial-mesenchymal transition in cancer cells.
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| 15. |
- Larsson, Chatarina, 1979-, et al.
(författare)
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Restoration of KMT2C/MLL3 in human colorectal cancer cells reinforces genome-wide H3K4me1 profiles and influences cell growth and gene expression
- 2020
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Ingår i: Clinical Epigenetics. - : Springer Nature. - 1868-7083 .- 1868-7075. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- Background The histone 3 lysine 4 (H3K4) monomethylase KMT2C is mutated across several cancer types; however, the effects of mutations on epigenome organization, gene expression, and cell growth are not clear. A frequently recurring mutation in colorectal cancer (CRC) with microsatellite instability is a single nucleotide deletion within the exon 38 poly-A(9) repeat (c.8390delA) which results in frameshift preceding the functional carboxy-terminal SET domain. To study effects ofKMT2Cexpression in CRC cells, we restored one allele to wild typeKMT2Cin the two CRC cell lines RKO and HCT116, which both are homozygous c.8390delA mutant. Results Gene editing resulted in increasedKMT2Cexpression, increased H3K4me1 levels, altered gene expression profiles, and subtle negative effects on cell growth, where higher dependence and stronger effects ofKMT2Cexpression were observed in RKO compared to HCT116 cells. Surprisingly, we found that the two RKO and HCT116 CRC cell lines have distinct baseline H3K4me1 epigenomic profiles. In RKO cells, a flatter genome-wide H3K4me1 profile was associated with more increased H3K4me1 deposition at enhancers, reduced cell growth, and more differential gene expression relative to HCT116 cells when KMT2C was restored. Profiling of H3K4me1 did not indicate a highly specific regulation of gene expression as KMT2C-induced H3K4me1 deposition was found globally and not at a specific enhancer sub-set in the engineered cells. Although we observed variation in differentially regulated gene sets between cell lines and individual clones, differentially expressed genes in both cell lines included genes linked to known cancer signaling pathways, estrogen response, hypoxia response, and aspects of immune system regulation. Conclusions Here, KMT2C restoration reduced CRC cell growth and reinforced genome-wide H3K4me1 deposition at enhancers; however, the effects varied depending upon the H3K4me1 status of KMT2C deficient cells. Results indicate that KMT2C inactivation may promote colorectal cancer development through transcriptional dysregulation in several pathways with known cancer relevance.
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| 16. |
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| 17. |
- Schoumans, Jacqueline, et al.
(författare)
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Comprehensive mutational analysis of a cohort of Swedish Cornelia de Lange syndrome patients
- 2007
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Ingår i: European Journal of Human Genetics. - : Springer Science and Business Media LLC. - 1018-4813 .- 1476-5438. ; 15:2, s. 143-149
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Tidskriftsartikel (refereegranskat)abstract
- Cornelia de Lange syndrome (CdLS; OMIM 122470) is a rare multiple congenital anomaly/mental retardation syndrome characterized by distinctive dysmorphic facial features, severe growth and developmental delay and abnormalities of the upper limbs. About 50% of CdLS patients have been found to have heterozygous mutations in the NIPBL gene and a few cases were recently found to be caused by mutations in the X-linked SMC1L1 gene. We performed a mutation screening of all NIPBL coding exons by direct sequencing in 11 patients (nine sporadic and two familial cases) diagnosed with CdLS in Sweden and detected mutations in seven of the cases. All were de novo, and six of the mutations have not been previously described. Four patients without identifiable NIPBL mutations were subsequently subjected to multiplex ligation-dependent probe amplification analysis to exclude whole exon deletions/duplications of NIPBL. In addition, mutation analysis of the 5' untranslated region (5' UTR) of NIPBL was performed. Tiling resolution array comparative genomic hybridization analysis was carried out on these four patients to detect cryptic chromosome imbalances and in addition the boys were screened for SMC1L1 mutations. We found a de novo 9p duplication with a size of 0.6 Mb in one of the patients with a CdLS-like phenotype but no mutations were detected in SMC1L1. So far, two genes (NIPBL and SMC1L1) have been identified causing CdLS or CdLS-like phenotypes. However, in a considerable proportion of individuals demonstrating the CdLS phenotype, mutations in any of these two genes are not found and other potential loci harboring additional CdLS-causing genes should be considered.
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| 18. |
- Stoimenov, Ivaylo, et al.
(författare)
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Computational and molecular tools for scalable rAAV mediated genome editing
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The rapid discovery of potential driver mutations through large scale mutational analyses of human cancers generates a need to characterize their cellular phenotypes. Among the techniques for genome editing, recombinant adeno-associated virus (rAAV) mediated gene targeting is particularly suited to knock-in of single nucleotide substitutions. However, the generation of gene targeting constructs and the targeting process is time consuming and labor-intense. To facilitate rAAV mediated gene targeting, we developed the first software and complementary automation friendly vector tools to generate optimized targeting constructs for editing human protein encoding genes. By computational approaches, rAAV constructs for editing ~72% of bases in protein-coding exons were designed. Similarly, ~81% of genes were predicted to be targetable by rAAV mediated knock-out. A Gateway based cloning system for facile generation of rAAV constructs suitable for robotic automation was developed and used in successful generation of targeting constructs. Together, these tools enable automated rAAV targeting construct design, generation as well as enrichment and expansion of targeted cells with desired integrations.
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| 19. |
- Sutton, LA, et al.
(författare)
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Comparative analysis of targeted next-generation sequencing panels for the detection of gene mutations in chronic lymphocytic leukemia: an ERIC multi-center study
- 2021
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Ingår i: Haematologica. - : Ferrata Storti Foundation (Haematologica). - 1592-8721 .- 0390-6078. ; 106:3, s. 682-691
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Tidskriftsartikel (refereegranskat)abstract
- Next-generation sequencing (NGS) has transitioned from research to clinical routine, yet the comparability of different technologies for mutation profiling remains an open question. We performed a European multicenter (n=6) evaluation of three amplicon-based NGS assays targeting 11 genes recurrently mutated in chronic lymphocytic leukemia. Each assay was assessed by two centers using 48 pre-characterized chronic lymphocytic leukemia samples; libraries were sequenced on the Illumina MiSeq instrument and bioinformatics analyses were centralized. Across all centers the median percentage of target reads ≥100x ranged from 94.2-99.8%. To rule out assay-specific technical variability, we first assessed variant calling at the individual assay level i.e. pairwise analysis of variants detected amongst partner centers. After filtering for variants present in the paired normal sample and removal of PCR/sequencing artefacts, the panels achieved 96.2% (Multiplicom), 97.7% (TruSeq) and 90% (HaloPlex) concordance at a VAF >0.5%. Reproducibility was assessed by looking at the inter-laboratory variation in detecting mutations and 107/115 (93% concordance) of mutations were detected by all 6 centers, while the remaining 8/115 (7%) variants were undetected by a single center and 6/8 of these variants concerned minor subclonal mutations (VAF <5%). We sought to investigate low-frequency mutations further by using a high-sensitivity assay containing unique molecular identifiers, which confirmed the presence of several minor subclonal mutations. Thus, while amplicon-based approaches can be adopted for somatic mutation detection with VAFs >5%, after rigorous validation, the use of unique molecular identifiers may be necessary to reach a higher sensitivity and ensure consistent and accurate detection of low-frequency variants.
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