The IMP3 RNA-binding protein is associated with metastasis and poor outcome in human cancer. Using solid cancer transcriptome data, we found that IMP3 correlates with HMGA2 mRNA expression. ...Cytoplasmic IMP3 granules contain HMGA2, and IMP3 dose-dependently increases HMGA2 mRNA. HMGA2 is regulated by let-7, and let-7 antagomiRs make HMGA2 refractory to IMP3. Removal of let-7 target sites eliminates IMP3-dependent stabilization, and IMP3-containing bodies are depleted of Ago1-4 and miRNAs. The relationship between Hmga2 mRNA and IMPs also exists in the developing limb bud, where IMP1-deficient embryos show dose-dependent Hmga2 mRNA downregulation. Finally, IMP3 ribonucleoproteins (RNPs) contain other let-7 target mRNAs, including LIN28B, and a global gene set enrichment analysis demonstrates that miRNA-regulated transcripts in general are upregulated following IMP3 induction. We conclude that IMP3 RNPs may function as cytoplasmic safe houses and prevent miRNA-directed mRNA decay of oncogenes during tumor progression.
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•IMP3 RNP granules contain large amounts of HMGA2 mRNA and other let-7 targets•IMP3 upregulates HMGA2 mRNA by opposing its interaction with Ago2/let-7•IMP3 RNP prevents miRNA-directed HMGA2 and LIN28B decay
The RNA-binding protein IMP3 is associated with metastasis and poor outcome in human cancer. Jønson et al. now show that IMP3 RNPs function as cytoplasmic safe houses in preventing miRNA-directed mRNA decay of oncogenes such as HMGA2 and LIN28B during cancer and development. This explains why poor outcome is a hallmark of IMP3-positive tumors and demonstrates how posttranscriptional events can be involved in tumor progression.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
ABSTRACT
Chromothripsis (CTH) is a phenomenon where multiple localized double‐stranded DNA breaks result in complex genomic rearrangements. Although the DNA‐repair mechanisms involved in CTH have ...been described, the mechanisms driving the localized “shattering” process remain unclear. High‐throughput sequence analysis of a familial germline CTH revealed an inserted SVAE retrotransposon associated with a 110‐kb deletion displaying hallmarks of L1‐mediated retrotransposition. Our analysis suggests that the SVAE insertion did not occur prior to or after, but concurrent with the CTH event. We also observed L1‐endonuclease potential target sites in other breakpoints. In addition, we found four Alu elements flanking the 110‐kb deletion and associated with an inversion. We suggest that chromatin looping mediated by homologous Alu elements may have brought distal DNA regions into close proximity facilitating DNA cleavage by catalytically active L1‐endonuclease. Our data provide the first evidence that active and inactive human retrotransposons can serve as endogenous mutagens driving CTH in the germline.
Although chromothripsis has been well described in cancer and some rare inherited disorders, the mutagens driving massive DNA rearrangement are unknown. Identification of a de novo L1‐mediated retrotransposition event, L1‐endonuclease cleavage sites at several breakpoint junctions, and Alu elements at inversion and deletion breakpoints implicate retrotransposons as drivers of a heritable chromothripsis event.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Background
Maternal immunization against KEL1 of the Kell blood group system can have serious adverse consequences for the fetus as well as the newborn baby. Therefore, it is important to determine ...the phenotype of the fetus to predict whether it is at risk. We present data that show the feasibility of predicting the fetal KEL1 phenotype using next‐generation sequencing (NGS) technology.
Study Design and Methods
The KEL1/2 single‐nucleotide polymorphism was polymerase chain reaction (PCR) amplified with one adjoining base, and the PCR product was sequenced using a genome analyzer (GAIIx, Illumina); several millions of PCR sequences were analyzed.
Results
The results demonstrated the feasibility of diagnosing the fetal KEL1 or KEL2 blood group from cell‐free DNA purified from maternal plasma.
Conclusion
This method requires only one primer pair, and the large amount of sequence information obtained allows well for statistical analysis of the data. This general approach can be integrated into current laboratory practice and has numerous applications. Besides DNA‐based predictions of blood group phenotypes, platelet phenotypes, or sickle cell anemia, and the determination of zygosity, various conditions of chimerism could also be examined using this approach. To our knowledge, this is the first report focused on antenatal blood group determination using NGS.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
We investigated a subject with an isolated cytochrome c oxidase (COX) deficiency presenting with an unusual phenotype characterised by neuropathy, exercise intolerance, obesity, and short stature.
...Blue-native polyacrylamide gel electrophoresis (BN-PAGE) analysis showed an almost complete lack of COX assembly in subject fibroblasts, consistent with the very low enzymatic activity, and pulse-labelling mitochondrial translation experiments showed a specific decrease in synthesis of the COX1 subunit, the core catalytic subunit that nucleates assembly of the holoenzyme. Whole exome sequencing identified compound heterozygous mutations (c.199dupC, c.215A>G) in COA3, a small inner membrane COX assembly factor, resulting in a pronounced decrease in the steady-state levels of COA3 protein. Retroviral expression of a wild-type COA3 cDNA completely rescued the COX assembly and mitochondrial translation defects, confirming the pathogenicity of the mutations, and resulted in increased steady-state levels of COX1 in control cells, demonstrating a role for COA3 in the stabilisation of this subunit. COA3 exists in an early COX assembly complex that contains COX1 and other COX assembly factors including COX14 (C12orf62), another single pass transmembrane protein that also plays a role in coupling COX1 synthesis with holoenzyme assembly. Immunoblot analysis showed that COX14 was undetectable in COA3 subject fibroblasts, and that COA3 was undetectable in fibroblasts from a COX14 subject, demonstrating the interdependence of these two COX assembly factors.
The mild clinical course in this patient contrasts with nearly all other cases of severe COX assembly defects that are usually fatal early in life, and underscores the marked tissue-specific involvement in mitochondrial diseases.
Oncofetal RNA‐binding IMPs have been implicated in mRNA localization, nuclear export, turnover and translational control. To depict the cellular actions of IMPs, we performed a loss‐of‐function ...analysis, which showed that IMPs are necessary for proper cell adhesion, cytoplasmic spreading and invadopodia formation. Loss of IMPs was associated with a coordinate downregulation of mRNAs encoding extracellular matrix and adhesion proteins. The transcripts were present in IMP RNP granules, implying that IMPs were directly involved in the post‐transcriptional control of the transcripts. In particular, we show that a 5.0 kb CD44 mRNA contained multiple IMP‐binding sites in its 3′UTR, and following IMP depletion this species became unstable. Direct knockdown of the CD44 transcript mimicked the effect of IMPs on invadopodia, and we infer that CD44 mRNA stabilization may be involved in IMP‐mediated invadopodia formation. Taken together, our results indicate that RNA‐binding proteins exert profound effects on cellular adhesion and invasion during development and cancer formation.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Inborn hemolytic anemia requiring frequent blood transfusions can be a life-threatening disease. Treatment, besides blood transfusion, includes iron chelation for prevention of iron accumulation due ...to frequent blood transfusions. We present the results of a clinical investigation where the proband was diagnosed with severe hemolytic anemia of unknown origin soon after birth. Transfusion was required every 4–6 weeks. After whole exome sequencing of the proband and his parents as well as a healthy sibling, we established that the proband had a compound heterozygous state carrying two rare variants in the erythrocytic spectrin gene, SPTA1. The maternal allele was a stop mutation (rs755630903) and the paternal allele was a missense mutation (rs375506528). The healthy sibling had the paternal variant but not the maternal variant. These rare variants of SPTA1 most likely account for the hemolytic anemia. A severely reduced osmotic resistance in the erythrocytes from the proband was demonstrated. Splenectomy considerably improved the hemolytic anemia and obviated the need for blood transfusion despite the severe clinical presentation.
Mutational screening of the breast cancer susceptibility gene BRCA1 leads to the identification of numerous pathogenic variants such as frameshift and nonsense variants, as well as large genomic ...rearrangements. The screening moreover identifies a large number of variants, for example, missense, silent, and intron variants, which are classified as variants of unknown clinical significance owing to the lack of causal evidence. Variants of unknown clinical significance can potentially have an impact on splicing and therefore functional examinations are warranted to classify whether these variants are pathogenic or benign. Here we validate a mini-gene splicing assay by comparing the results of 24 variants with previously published data from RT-PCR analysis on RNA from blood samples/lymphoblastoid cell lines. The analysis showed an overall concordance of 100%. In addition, we investigated 13 BRCA1 variants of unknown clinical significance or putative variants affecting splicing by in silico analysis and mini-gene splicing assay. Both the in silico analysis and mini-gene splicing assay classified six BRCA1 variants as pathogenic (c.80+1G>A, c.132C>T (p.=), c.213-1G>A, c.670+1delG, c.4185+1G>A, and c.5075-1G>C), whereas six BRCA1 variants were classified as neutral (c.-19-22_-19-21dupAT, c.302-15C>G, c.547+14delG, c.4676-20A>G, c.4987-21G>T, and c.5278-14C>G) and one BRCA1 variant remained unclassified (c.670+16G>A). In conclusion, our study emphasizes that in silico analysis and mini-gene splicing assays are important for the classification of variants, especially if no RNA is available from the patient. This knowledge is crucial for proper genetic counseling of patients and their family members.
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, SIK, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Genetic testing of BRCA1/2 includes screening for single nucleotide variants and small insertions/deletions and for larger copy number variations (CNVs), primarily by Sanger sequencing and multiplex ...ligation-dependent probe amplification (MLPA). With the advent of next-generation sequencing (NGS), it has become feasible to provide CNV information and sequence data using a single platform. We report the use of NGS gene panel sequencing on the Illumina MiSeq platform and JSI SeqPilot SeqNext software to call germline CNVs in BRCA1 and BRCA2. For validation 18 different BRCA1/BRCA2 CNVs previously identified by MLPA in 48 Danish breast and/or ovarian cancer families were analyzed. Moreover, 120 patient samples previously determined as negative for BRCA1/BRCA2 CNVs by MLPA were included in the analysis. Comparison of the NGS data with the data from MLPA revealed that the sensitivity was 100%, whereas the specificity was 95%. Taken together, this study validates a one-step bioinformatics work-flow to call germline BRCA1/2 CNVs using data obtained by NGS of a breast cancer gene panel. The work-flow represents a robust and easy-to-use method for full BRCA1/2 screening, which can be easily implemented in routine diagnostic testing and adapted to genes other than BRCA1/2.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Germ-line mutations in the
RAD51C
gene have recently been identified in families with breast and ovarian cancer and have been associated with an increased risk of ovarian cancer. In this study, we ...describe the frequency of pathogenic
RAD51C
mutations identified in Danish breast and/or ovarian cancer families. We screened the
RAD51C
gene in 1228 Danish hereditary breast and/or ovarian cancer families by next-generation sequencing analysis. The frequency of the identified variants was examined in the exome sequencing project database and in data from 2000 Danish exomes and the presumed significance of missense and intronic variants was predicted by in silico analysis. We identified six families with a pathogenic mutation in
RAD51C
, including three frameshift mutations, one nonsense mutation, and 2 missense mutations. Overall, pathogenic
RAD51C
mutations were identified in 0.5 % of Danish families with increased risk of hereditary breast and/or ovarian cancer. Moreover, we identified 24 additional
RAD51C
variants of which 14 have not been previously reported in the literature. In this study, we determine the prevalence of
RAD51C
mutations in Danish breast and/or ovarian cancer families. We identified six pathogenic
RAD51C
mutations as well as 23 variants of uncertain clinical significance and one benign variant. Together, the study extends our knowledge of the
RAD51C
mutation spectrum and supports that
RAD51C
should be included in gene panel testing of individuals with high risk of breast and ovarian cancer.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
10.
Personalized oncology: genomic screening in phase 1 Tuxen, Ida Viller; Jønson, Lars; Santoni-Rugiu, Eric ...
APMIS : acta pathologica, microbiologica et immunologica Scandinavica,
August 2014, Volume:
122, Issue:
8
Journal Article
Peer reviewed
Improvements in cancer genomics and tumor biology have reinforced the evidence of cancer development driven by numerous genomic alterations. Advanced genomics technology can be used to characterize ...genomic alterations that potentially drive tumor growth. With the possibility of screening thousands of genes simultaneously, personalized molecular medicine has become an option. New treatments are being investigated in phase 1 trials around the world. Traditionally, the goal of phase 1 studies was to determine the optimal dose and assess dose‐limiting toxicity of a potential new experimental drug. Only a limited number of patients will benefit from the treatment. However, introducing genomic mapping to select patients for early clinical trials with targeted molecular therapy according to the genomic findings, may lead to a better outcome for the patient, an enrichment of phase 1 trials, and thereby accelerated drug development. The overall advantage is to determine which mutation profiles correlate with sensitivity or lack of resistance to specific targeted therapies. The utility and current limitations of genomic screening to guide selection to Phase 1 clinical trial will be discussed.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
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