Publications by authors named "Peter Mouritzen"

30 Publications

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Verification of CRISPR editing and finding transgenic inserts by Xdrop™ Indirect sequence capture followed by short- and long- read sequencing.

Methods 2021 Feb 11. Epub 2021 Feb 11.

Samplix ApS, Mileparken 28, Herlev, Denmark. Electronic address:

Validation of CRISPR-Cas9 editing typically explore the immediate vicinity of the gene editing site and distal off-target sequences, which have led to the conclusion that CRISPR-Cas9 editing is very specific. However, an increasing number of studies suggest that on-target unintended editing events like deletions and insertions are relatively frequent but unfortunately often missed in the validation of CRISPR-Cas9 editing. The deletions may be several kilobases-long and only affect one allele. The gold standard in molecular validation of gene editing is direct sequencing of relatively short PCR amplicons. This approach allows the detection of small editing events but fails in detecting large rearrangements, in particular when only one allele is affected. Detection of large rearrangements requires that an extended region is analyzed and the characterization of events may benefit from long-read sequencing. Here we implemented Xdrop™, a new microfluidic technology that allows targeted enrichment of long regions (∼ 100 kb) using just a single standard PCR primer set. Sequencing of the enriched CRISPR-Cas9 gene edited region in 4 cell lines on long- and short- read sequencing platforms unravelled unknown and unintended genome editing events. The analysis revealed accidental kb large insertions in 3 of the cell lines, which remained undetected using standard procedures. We also applied the targeted enrichment approach to identify the integration site of a transgene in a mouse line. The results demonstrate the potential of this technology in gene editing validation as well as in more classic transgenics.
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http://dx.doi.org/10.1016/j.ymeth.2021.02.003DOI Listing
February 2021

A genome-wide screen reveals microRNAs in peripheral sensory neurons driving painful diabetic neuropathy.

Pain 2020 Dec 30. Epub 2020 Dec 30.

Department of Molecular Pharmacology, Pharmacology Institute, Medical Faculty Heidelberg, Heidelberg University, Heidelberg, Germany. Dr. Bali is now with the Department of Experimental Pain Research, Mannheim Center for Translational Neuroscience (MCTN), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany EMBL Rome, Monterotondo, Italy Exiqon A/S, Vedbaek, Denmark Department of Experimental Pain Research, Mannheim Center for Translational Neuroscience (MCTN), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany.

Abstract: Diabetes is a leading cause of peripheral neuropathy (diabetic peripheral neuropathy, DPN), and uncontrolled long-lasting hyperglycemia leads to severe complications. A major proportion of diabetics develop excruciating pain with a variable course. Mechanisms leading to painful DPN are not completely understood and treatment options limited. We hypothesized that epigenetic modulation at the level of microRNA (miRNA) expression triggered by metabolic imbalance and nerve damage regulates the course of pain development. We used clinically relevant preclinical models, genome-wide screening, in silico analyses, cellular assays, miRNA fluorescent in situ hybridization, in vivo molecular manipulations, and behavioral analyses in the current study. We identified miRNAs and their targets that critically impact on nociceptive hypersensitivity in painful DPN. Our analyses identify miR-33 and miR-380 expressed in nociceptive neurons as critical denominators of diabetic pain and miR-124-1 as a mediator of physiological nociception. Our comprehensive analyses on the putative mRNA targets for miR-33 or miR-124-1 identified a set of mRNAs that are regulated after miR-33 or miR-124-1 overexpression in dorsal root ganglia in vivo. Our results shed light on the regulation of DPN pathophysiology and implicate specific miRNAs as novel therapeutic targets for treating painful DPN.
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http://dx.doi.org/10.1097/j.pain.0000000000002159DOI Listing
December 2020

Validation of the four-miRNA biomarker panel MiCaP for prediction of long-term prostate cancer outcome.

Sci Rep 2020 07 1;10(1):10704. Epub 2020 Jul 1.

Department of Molecular Medicine (MOMA), Aarhus University Hospital, Aarhus, Denmark.

Improved prostate cancer prognostic biomarkers are urgently needed. We previously identified the four-miRNA prognostic biomarker panel MiCaP ((miR-23a-3p × miR-10b-5p)/(miR-133a-3p × miR-374b-5p)) for prediction of biochemical recurrence (BCR) after radical prostatectomy (RP). Here, we identified an optimal numerical cut-off for MiCaP dichotomisation using a training cohort of 475 RP patients and tested this in an independent cohort of 281 RP patients (PCA281). Kaplan-Meier, uni- and multivariate Cox regression analyses were conducted for multiple endpoints: BCR, metastatic-(mPC) and castration-resistant prostate cancer (CRPC), prostate cancer-specific (PCSS) and overall survival (OS). Functional effects of the four MiCaP miRNAs were assessed by overexpression and inhibition experiments in prostate cancer cell lines. We found the numerical value 5.709 optimal for MiCaP dichotomisation. This was independently validated in PCA281, where a high MiCaP score significantly [and independent of the Cancer of the Prostate Risk Assessment Postsurgical (CAPRA-S) score] predicted BCR, progression to mPC and CRPC, and PCSS, but not OS. Harrell's C-index increased upon addition of MiCaP to CAPRA-S for all endpoints. Inhibition of miR-23a-3p and miR-10b-5p, and overexpression of miR-133a-3p and miR-374b-5p significantly reduced cell survival. Our results may promote future implementation of a MiCaP-based test for improved prostate cancer risk stratification.
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http://dx.doi.org/10.1038/s41598-020-67320-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7329825PMC
July 2020

Profiling of Circulating microRNAs in Prostate Cancer Reveals Diagnostic Biomarker Potential.

Diagnostics (Basel) 2020 Mar 28;10(4). Epub 2020 Mar 28.

Department of Molecular Medicine, Aarhus University Hospital, 8200 Aarhus N, Denmark.

Early detection of prostate cancer (PC) is paramount as localized disease is generally curable, while metastatic PC is generally incurable. There is a need for improved, minimally invasive biomarkers as current diagnostic tools are inaccurate, leading to extensive overtreatment while still missing some clinically significant cancers. Consequently, we profiled the expression levels of 92 selected microRNAs by RT-qPCR in plasma samples from 753 patients, representing multiple stages of PC and non-cancer controls. First, we compared plasma miRNA levels in patients with benign prostatic hyperplasia (BPH) or localized prostate cancer (LPC), versus advanced prostate cancer (APC). We identified several dysregulated microRNAs with a large overlap of 59 up/down-regulated microRNAs between BPH versus APC and LPC versus APC. Besides identifying several novel PC-associated dysregulated microRNAs in plasma, we confirmed the previously reported upregulation of miR-375 and downregulation of miR-146a-5p. Next, by randomly splitting our dataset into a training and test set, we identified and successfully validated a novel four microRNA diagnostic ratio model, termed (miR-375*miR-33a-5p/miR-16-5p*miR-409-3p). Combined in a model with prostate specific antigen (PSA), digital rectal examination status, and age, predicted the outcomes of transrectal ultrasound (TRUS)-guided biopsies (negative vs. positive) with greater accuracy than PSA alone (Training: area under the curve (AUC), model = 0.84; AUC, PSA = 0.63. Test set: AUC, model = 0.67; AUC, PSA = 0.56). It may be possible in the future to use this simple and minimally invasive test in combination with existing clinical parameters for a more accurate selection of patients for prostate biopsy.
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http://dx.doi.org/10.3390/diagnostics10040188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7235761PMC
March 2020

Novel DNA methylation biomarkers show high sensitivity and specificity for blood-based detection of colorectal cancer-a clinical biomarker discovery and validation study.

Clin Epigenetics 2019 11 14;11(1):158. Epub 2019 Nov 14.

Department of Molecular Medicine, Aarhus University Hospital, Palle Juul-Jensens Boulevard 99, 8200, Aarhus N, Denmark.

Background: Early detection plays an essential role to reduce colorectal cancer (CRC) mortality. While current screening methods suffer from poor compliance, liquid biopsy-based strategies for cancer detection is rapidly gaining promise. Here, we describe the development of TriMeth, a minimal-invasive blood-based test for detection of early-stage colorectal cancer. The test is based on assessment of three tumour-specific DNA methylation markers in circulating cell-free DNA.

Results: A thorough multi-step biomarker discovery study based on DNA methylation profiles of more than 5000 tumours and blood cell populations identified CRC-specific DNA methylation markers. The DNA methylation patterns of biomarker candidates were validated by bisulfite sequencing and methylation-specific droplet digital PCR in CRC tumour tissue and peripheral blood leucocytes. The three best performing markers were first applied to plasma from 113 primarily early-stage CRC patients and 87 age- and gender-matched colonoscopy-verified controls. Based on this, the test scoring algorithm was locked, and then TriMeth was validated in an independent cohort comprising 143 CRC patients and 91 controls. Three DNA methylation markers, C9orf50, KCNQ5, and CLIP4, were identified, each capable of discriminating plasma from colorectal cancer patients and healthy individuals (areas under the curve 0.86, 0.91, and 0.88). When combined in the TriMeth test, an average sensitivity of 85% (218/256) was observed (stage I: 80% (33/41), stage II: 85% (121/143), stage III: 89% (49/55), and stage IV: 88% (15/17)) at 99% (176/178) specificity in two independent plasma cohorts.

Conclusion: TriMeth enables detection of early-stage colorectal cancer with high sensitivity and specificity. The reported results underline the potential utility of DNA methylation-based detection of circulating tumour DNA in the clinical management of colorectal cancer.
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http://dx.doi.org/10.1186/s13148-019-0757-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6854894PMC
November 2019

Elevated miR-615-3p Expression Predicts Adverse Clinical Outcome and Promotes Proliferation and Migration of Prostate Cancer Cells.

Am J Pathol 2019 12 17;189(12):2377-2388. Epub 2019 Sep 17.

Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark; Department of Clinical Medicine, Aarhus University, Aarhus, Denmark. Electronic address:

miR-615-3p has previously been described as up-regulated in prostate cancer (PC) tissue samples compared with nonmalignant controls; however, its prognostic potential and functional role in PC remain largely unknown. In this study, we investigated the clinical and biological relevance of miR-615-3p in PC. The expression of miR-615-3p was measured in PC tissue specimens from 239 men who underwent radical prostatectomy (RP), and it was investigated if miR-615-3p could predict postoperative biochemical recurrence (BCR). These findings were subsequently validated in three independent RP cohorts (n = 222, n = 273, and n = 387) and functional overexpression studies conducted in PC cells (PC3M). High miR-615-3p expression was significantly associated with BCR in four independent PC patient cohorts (P < 0.05, log-rank test). In addition, high miR-615-3p expression was a significant predictor of PC-specific survival in univariate (hazard ratio, 3.75; P < 0.001) and multivariate (hazard ratio, 2.66; P = 0.008) analysis after adjustment for the Cancer of the Prostate Risk Assessment Post-Surgical (CAPRA-S) nomogram in a merged RP cohort (n = 734). Moreover, overexpression of miR-615-3p in PC cells (PC3M) significantly increased cell viability, proliferation, apoptosis, and migration. Together, our results suggest that miR-615-3p is a significant predictor of postoperative BCR and PC-specific survival and has oncogenic functions in PC cells.
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http://dx.doi.org/10.1016/j.ajpath.2019.08.007DOI Listing
December 2019

A novel combined miRNA and methylation marker panel (miMe) for prediction of prostate cancer outcome after radical prostatectomy.

Int J Cancer 2019 12 7;145(12):3445-3452. Epub 2019 Jun 7.

Department of Molecular Medicine (MOMA), Aarhus University Hospital, Aarhus, Denmark.

Improved prognostic biomarkers are needed to guide personalized prostate cancer (PC) treatment decisions. Due to the prominent molecular heterogeneity of PC, multimarker panels may be more robust. Here, 25 selected top-candidate miRNA and methylation markers for PC were profiled by qPCR in malignant radical prostatectomy (RP) tissue specimens from 198 PC patients (Cohort 1, training). Using GLMnet, we trained a novel multimarker model (miMe) comprising nine miRNAs and three methylation markers that predicted postoperative biochemical recurrence (BCR) independently of the established clinicopathological CAPRA-S nomogram in Cox multivariate regression analysis in Cohort 1 (HR [95% CI]: 1.53 [1.26-1.84], p < 0.001). This result was successfully validated in two independent RP cohorts (Cohort 2, n = 159: HR [95% CI]: 1.35 [1.06-1.73], p = 0.015. TCGA, n = 350: HR [95% CI]: 1.34 [1.01-1.77], p = 0.04). Notably, in CAPRA-S low-risk patients, a high miMe score was associated with >6 times higher risk of BCR, suggesting that miMe may help identify PC patients at high risk of progression despite favorable clinicopathological factors postsurgery. Finally, miMe was a significant predictor of cancer-specific survival (p = 0.019, log-rank test) in a merged analysis of 357 RP patients. In conclusion, we trained, tested and validated a novel 12-marker panel (miMe) that showed significant independent prognostic value in three RP cohorts. In the future, combining miMe score with existing clinical nomograms may improve PC risk stratification and thus help guide treatment decisions.
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http://dx.doi.org/10.1002/ijc.32427DOI Listing
December 2019

A five-microRNA model (pCaP) for predicting prostate cancer aggressiveness using cell-free urine.

Int J Cancer 2019 11 8;145(9):2558-2567. Epub 2019 Apr 8.

Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark.

Improved biomarkers for prostate cancer (PC) risk stratification are urgently needed. Here, we aimed to develop a novel multimarker model for prediction of biochemical recurrence (BCR) after curatively intended radical prostatectomy (RP), based on minimally invasive sampling of blood and urine. We initially measured the levels of 45 selected miRNAs by RT-qPCR in exosome enriched cell-free urine samples collected prior to RP from 215 PC patients (Cohort 1, training). We trained a novel logistic regression model (pCaP), comprising five urine miRNAs (miR-151a-5p, miR-204-5p, miR-222-3p, miR-23b-3p and miR-331-3p) and serum prostate-specific antigen (PSA), which significantly predicted time to BCR in Cohort 1 (univariate Cox regression analysis: HR = 3.12, p < 0.001). Next, using the same exact numeric cutoff for dichotomization as trained in Cohort 1, we tested and successfully validated the prognostic potential of pCaP in two additional cohorts, including 199 (Cohort 2, HR = 2.24, p = 0.002) and 205 (Cohort 3, HR = 2.15, p = 0.004) RP patients, respectively. pCaP remained a significant predictor of BCR, also after adjustment for pathological T-stage, surgical margin status and Gleason grade group (p < 0.05 in multivariate Cox regression analysis: HR = 2.72, 1.94 and 1.83 for Cohorts 1, 2 and 3, respectively). Additionally, pCaP scores correlated positively with the established clinical risk stratification nomogram CAPRA in all three PC cohorts (Pearson's rho: 0.45, 0.39 and 0.44). Together, our results suggest that the minimally invasive pCaP model could potentially be used in the future to improve PC risk stratification and to guide more personalized treatment decisions. Further clinical validation studies are warranted.
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http://dx.doi.org/10.1002/ijc.32296DOI Listing
November 2019

Aberrant , , and Hypermethylation has Potential as a Prognostic Biomarker for Prostate Cancer.

Int J Mol Sci 2019 Mar 7;20(5). Epub 2019 Mar 7.

Department of Molecular Medicine (MOMA), Aarhus University Hospital (AUH), Palle Juul-Jensens Boulevard 99, 8200 Aarhus N, Denmark.

Prostate cancer (PCa) is a clinically heterogeneous disease and currently, accurate diagnostic and prognostic molecular biomarkers are lacking. This study aimed to identify novel DNA hypermethylation markers for PCa with future potential for blood-based testing. Accordingly, to search for genes specifically hypermethylated in PCa tissue samples and not in blood cells or other cancer tissue types, we performed a systematic analysis of genome-wide DNA methylation data (Infinium 450K array) available in the Marmal-aid database for 4072 malignant/normal tissue samples of various types. We identified eight top candidate markers (cg12799885, , , , , , , and ) that were specifically hypermethylated in PCa tissue samples and hypomethylated in other benign and malignant tissue types, including in peripheral blood cells. Potential as diagnostic and prognostic biomarkers was further assessed by the quantitative methylation specific PCR (qMSP) analysis of 37 nonmalignant and 197 PCa tissue samples from an independent population. Here, all eight hypermethylated candidates showed high sensitivity (75⁻94%) and specificity (84⁻100%) for PCa. Furthermore, , , and hypermethylation was significantly associated with biochemical recurrence (BCR) after radical prostatectomy (RP; 197 patients), independent of the routine clinicopathological variables. is the most promising single candidate marker (hazard ratio (HR) (95% confidence interval (CI)): 1.96 (1.24⁻3.10), adjusted = 0.016; multivariate cox regression). Further validation studies are warranted and should investigate the potential value of these hypermethylation candidate markers for blood-based testing also.
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http://dx.doi.org/10.3390/ijms20051173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6429171PMC
March 2019

Independent Validation of a Diagnostic Noninvasive 3-MicroRNA Ratio Model () for Prostate Cancer in Cell-Free Urine.

Clin Chem 2019 04 6;65(4):540-548. Epub 2019 Feb 6.

Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark;

Background: Detection of prostate cancer (PC) based on serum prostate-specific antigen (PSA) testing leads to many unnecessary prostate biopsies, overdiagnosis, and overtreatment of clinically insignificant tumors. Thus, novel and more accurate molecular biomarkers are required.

Methods: Using reverse transcription quantitative PCR, we measured the concentrations of 45 preselected microRNAs (miRNAs) in extracellular vesicle-enriched cell-free urine samples from 4 independent patient cohorts from Spain and Denmark, including 758 patients with clinically localized PC, 289 noncancer controls with benign prostatic hyperplasia (BPH), and 233 patients undergoing initial transrectal ultrasound (TRUS)-guided prostate biopsy owing to PC suspicion (101 with benign and 132 with malignant outcome). Diagnostic potential was assessed by ROC and decision curve analysis.

Results: We identified and successfully validated 8 upregulated and 21 downregulated miRNAs in urine from PC patients. Furthermore, we validated a previously identified 3-miRNA diagnostic ratio model, (miR-222-3p*miR-24-3p/miR-30c-5p). High scores were distinctive of PC in urine samples from BPH vs PC patients in 3 independent cohorts [area under the curve (AUC) = 0.84, 0.71, 0.72]. Additionally, predicted TRUS biopsy results with greater accuracy than PSA (AUC = 0.644; AUC PSA = 0.527) for patients within the diagnostic gray zone (PSA ≤ 10 ng/mL).

Conclusions: We successfully validated a urine-based diagnostic 3-miRNA signature for PC () in 3 independent patient cohorts from 2 countries. In the future, the simple and noninvasive test may be used to help more accurately select patients for prostate biopsy. Prospective clinical validation is warranted.
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http://dx.doi.org/10.1373/clinchem.2018.296681DOI Listing
April 2019

Analysis of a gene panel for targeted sequencing of colorectal cancer samples.

Oncotarget 2018 Feb 10;9(10):9043-9060. Epub 2018 Jan 10.

Oncology Department, Vejle Hospital, Vejle 7100, Denmark.

Colorectal cancer (CRC) is a leading cause of death worldwide. Surgical intervention is a successful treatment for stage I patients, whereas other more advanced cases may require adjuvant chemotherapy. The selection of effective adjuvant treatments remains, however, challenging. Accurate patient stratification is necessary for the identification of the subset of patients likely responding to treatment, while sparing others from pernicious treatment. Targeted sequencing approaches may help in this regard, enabling rapid genetic investigation, and at the same time easily applicable in routine diagnosis. We propose a set of guidelines for the identification, including variant calling and filtering, of somatic mutations driving tumorigenesis in the absence of matched healthy tissue. We also discuss the inclusion criteria for the generation of our gene panel. Furthermore, we evaluate the prognostic impact of individual genes, using Cox regression models in the context of overall survival and disease-free survival. These analyses confirmed the role of commonly used biomarkers, and shed light on controversial genes such as . Applying those guidelines, we created a novel gene panel to investigate the onset and progression of CRC in 273 patients. Our comprehensive biomarker set includes 266 genes that may play a role in the progression through the different stages of the disease. Tracing the developmental state of the tumour, and its resistances, is instrumental in patient stratification and reliable decision making in precision clinical practice.
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http://dx.doi.org/10.18632/oncotarget.24138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5823670PMC
February 2018

Two approaches for estimating the lower limit of quantitation (LLOQ) of microRNA levels assayed as exploratory biomarkers by RT-qPCR.

BMC Biotechnol 2018 02 2;18(1). Epub 2018 Feb 2.

Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD, 20993, USA.

Background: Circulating microRNAs are undergoing exploratory use as safety biomarkers in drug development. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is one common approach used to quantitate levels of microRNAs in samples that includes the use of a standard curve of calibrators fit to a regression model. Guidelines are needed for setting assay quantitation thresholds that are appropriate for this method and to biomarker pre-validation.

Results: In this report, we develop two workflows for determining a lower limit of quantitation (LLOQ) for RT-qPCR assays of microRNAs in exploratory studies. One workflow is based on an error threshold calculated by a logistic model of the calibration curve data. The second workflow is based on a threshold set by the sample blank, which is the no template control for RT-qPCR. The two workflows are used to set lower thresholds of reportable microRNA levels for an example dataset in which miR-208a levels in biofluids are quantitated in a cardiac injury model. LLOQ thresholds set by either workflow are effective in filtering out microRNA values with large uncertainty estimates.

Conclusions: Two workflows for LLOQ determinations are presented in this report that provide methods that are easy to implement in investigational studies of microRNA safety biomarkers and offer choices in levels of conservatism in setting lower limits of acceptable values that facilitate interpretation of results.
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http://dx.doi.org/10.1186/s12896-018-0415-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5796571PMC
February 2018

Exosomal cargo including microRNA regulates sensory neuron to macrophage communication after nerve trauma.

Nat Commun 2017 11 24;8(1):1778. Epub 2017 Nov 24.

Wolfson Centre for Age Related Diseases, King's College London, London, SE1 1UL, UK.

Following peripheral axon injury, dysregulation of non-coding microRNAs (miRs) occurs in dorsal root ganglia (DRG) sensory neurons. Here we show that DRG neuron cell bodies release extracellular vesicles, including exosomes containing miRs, upon activity. We demonstrate that miR-21-5p is released in the exosomal fraction of cultured DRG following capsaicin activation of TRPV1 receptors. Pure sensory neuron-derived exosomes released by capsaicin are readily phagocytosed by macrophages in which an increase in miR-21-5p expression promotes a pro-inflammatory phenotype. After nerve injury in mice, miR-21-5p is upregulated in DRG neurons and both intrathecal delivery of a miR-21-5p antagomir and conditional deletion of miR-21 in sensory neurons reduce neuropathic hypersensitivity as well as the extent of inflammatory macrophage recruitment in the DRG. We suggest that upregulation and release of miR-21 contribute to sensory neuron-macrophage communication after damage to the peripheral nerve.
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http://dx.doi.org/10.1038/s41467-017-01841-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5701122PMC
November 2017

Diagnostic and Prognostic MicroRNA Biomarkers for Prostate Cancer in Cell-free Urine.

Eur Urol Focus 2018 12 9;4(6):825-833. Epub 2017 Mar 9.

Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark. Electronic address:

Background: Widespread use of prostate-specific antigen (PSA) testing for prostate cancer (PC) detection has led to extensive overdiagnosis and overtreatment. Urine-based microRNA (miRNA) biomarkers could be useful in PC diagnosis and prognosis.

Objective: To train and validate urine-based microRNA (miRNA) biomarkers that may assist in PC diagnosis and prognosis.

Design, Setting, And Participants: We profiled the expression levels of 92 miRNAs via reverse transcriptase-poymerase chain reaction in cell-free urine samples from 29 patients with benign prostatic hyperplasia (BPH) and 215 patients with clinically localized PC (cohort 1). Our findings were validated in an independent cohort of 29 BPH patients and 220 patients with clinically localized PC (cohort 2).

Results And Limitations: We identified and validated several deregulated miRNAs in urine samples from PC patients. In addition, we trained a novel diagnostic three-miRNA model (miR-222-3p*miR-24-3p/miR-30c-5p) that distinguished BPH and PC patients with an area under the curve (AUC) of 0.95 in cohort 1, and was successfully validated in cohort 2 (AUC 0.89). Furthermore, we trained a novel prognostic three-miRNA model (miR-125b-5p*let-7a-5p/miR-151-5p) that predicted time to biochemical recurrence after radical prostatectomy independently of routine clinicopathological parameters in cohort 1, and was successfully validated in cohort 2.

Conclusions: Future clinical implementation of our novel diagnostic and prognostic three-miRNA signatures could help in primary diagnosis of PC and guide treatment decisions. Further validation studies are warranted.

Patient Summary: Using two large patient cohorts, we searched for novel prostate cancer biomarkers in urine. We found two new sets of microRNA biomarkers in urine that could accurately predict the presence of prostate cancer and the likelihood of recurrence after prostatectomy. Further studies are needed before an actual clinical test can be developed.
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http://dx.doi.org/10.1016/j.euf.2017.02.018DOI Listing
December 2018

Quantitative RT-PCR for MicroRNAs in Biofluids.

Methods Mol Biol 2017 ;1641:379-398

Exiqon A/S, Skelstedet 16, Vedbæk, DK-2950, Denmark.

MicroRNAs in biofluids hold great promise as minimally invasive diagnostic biomarkers for a wide range of diseases and biological processes. One of the most sensitive technologies for detection and measuring expression levels of microRNA is quantitative RT-PCR. However, quantification of microRNA in biofluid samples is challenging in many ways. Biofluids contain low levels of RNA and high levels of inhibitors of enzymatic processes like reverse transcription and PCR. Furthermore, biofluids are susceptible to many preanalytical variables. Here we describe procedures developed to address these challenges, which include highly sensitive and accurate microRNA detection methods, combined with optimized protocols for sample handling and preparation, and extensive quality control (QC) procedures.
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http://dx.doi.org/10.1007/978-1-4939-7172-5_21DOI Listing
April 2018

Clinical Implications of Monitoring Circulating Tumor DNA in Patients with Colorectal Cancer.

Clin Cancer Res 2017 Sep 9;23(18):5437-5445. Epub 2017 Jun 9.

Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark.

We investigated whether detection of ctDNA after resection of colorectal cancer identifies the patients with the highest risk of relapse and, furthermore, whether longitudinal ctDNA analysis allows early detection of relapse and informs about response to intervention. In this longitudinal cohort study, we used massively parallel sequencing to identify somatic mutations and used these as ctDNA markers to detect minimal residual disease and to monitor changes in tumor burden during a 3-year follow-up period. A total of 45 patients and 371 plasma samples were included. Longitudinal samples from 27 patients revealed ctDNA postoperatively in all relapsing patients ( = 14), but not in any of the nonrelapsing patients. ctDNA detected relapse with an average lead time of 9.4 months compared with CT imaging. Of 21 patients treated for localized disease, six had ctDNA detected within 3 months after surgery. All six later relapsed compared with four of the remaining patients [HR, 37.7; 95% confidence interval (CI), 4.2-335.5; < 0.001]. The ability of a 3-month ctDNA analysis to predict relapse was confirmed in 23 liver metastasis patients (HR 4.9; 95% CI, 1.5-15.7; = 0.007). Changes in ctDNA levels induced by relapse intervention ( = 19) showed good agreement with changes in tumor volume (κ = 0.41; Spearman = 0.4). Postoperative ctDNA detection provides evidence of residual disease and identifies patients at very high risk of relapse. Longitudinal surveillance enables early detection of relapse and informs about response to intervention. These observations have implications for the postoperative management of colorectal cancer patients. .
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http://dx.doi.org/10.1158/1078-0432.CCR-17-0510DOI Listing
September 2017

Genome-Wide Comparison of Next-Generation Sequencing and qPCR Platforms for microRNA Profiling in Serum.

Methods Mol Biol 2017 ;1580:21-44

Exiqon A/S, Vedbaek, Denmark.

This study compares next-generation sequencing (NGS) technologies that have been optimized specifically for biofluid samples, with more established qPCR-based methods for profiling microRNAs in biofluids. The same patient serum samples were analyzed by NGS and qPCR, and differences in the serum microRNA profile between HBV and HCV infected patients were investigated. While there was overall good agreement between NGS and qPCR, there were some differences between the platforms, highlighting the importance of validation.
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http://dx.doi.org/10.1007/978-1-4939-6866-4_3DOI Listing
February 2018

Absolute Measurement of Cardiac Injury-Induced microRNAs in Biofluids across Multiple Test Sites.

Toxicol Sci 2016 11 7;154(1):115-125. Epub 2016 Sep 7.

SAS Institute Inc, Cary, North Carolina 27513.

Extracellular microRNAs (miRNAs) represent a promising new source of toxicity biomarkers that are sensitive indicators of site of tissue injury. In order to establish reliable approaches for use in biomarker validation studies, the HESI technical committee on genomics initiated a multi-site study to assess sources of variance associated with quantitating levels of cardiac injury induced miRNAs in biofluids using RT-qPCR. Samples were generated at a central site using a model of acute cardiac injury induced in male Wistar rats by 0.5 mg/kg isoproterenol. Biofluid samples were sent to 11 sites for measurement of 3 cardiac enriched miRNAs (miR-1-3p, miR-208a-3p, and miR-499-5p) and 1 miRNA abundant in blood (miR-16-5p) or urine (miR-192-5p) by absolute quantification using calibration curves of synthetic miRNAs. The samples included serum and plasma prepared from blood collected at 4 h, urine collected from 6 to 24 h, and plasma prepared from blood collected at 24 h post subcutaneous injection. A 3 parameter logistic model was utilized to fit the calibration curve data and estimate levels of miRNAs in biofluid samples by inverse prediction. Most sites observed increased circulating levels of miR-1-3p and miR-208a-3p at 4 and 24 h after isoproterenol treatment, with no difference seen between serum and plasma. The biological differences in miRNA levels and sample type dominated as sources of variance, along with outlying performance by a few sites. The standard protocol established in this study was successfully implemented across multiple sites and provides a benchmark method for further improvements in quantitative assays for circulating miRNAs.
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http://dx.doi.org/10.1093/toxsci/kfw143DOI Listing
November 2016

miRNA profiling of circulating EpCAM(+) extracellular vesicles: promising biomarkers of colorectal cancer.

J Extracell Vesicles 2016 29;5:31488. Epub 2016 Aug 29.

Department of Molecular Medicine (MOMA), Aarhus University Hospital, Aarhus, Denmark;

Cancer cells secrete small membranous extracellular vesicles (EVs) into their microenvironment and circulation. These contain biomolecules, including proteins and microRNAs (miRNAs). Both circulating EVs and miRNAs have received much attention as biomarker candidates for non-invasive diagnostics. Here we describe a sensitive analytical method for isolation and subsequent miRNA profiling of epithelial-derived EVs from blood samples of patients with colorectal cancer (CRC). The epithelial-derived EVs were isolated by immunoaffinity-capture using the epithelial cell adhesion molecule (EpCAM) as marker. This approach mitigates some of the specificity issues observed in earlier studies of circulating miRNAs, in particular the negative influence of miRNAs released by erythrocytes, platelets and non-epithelial cells. By applying this method to 2 small-scale patient cohorts, we showed that blood plasma isolated from CRC patients prior to surgery contained elevated levels of 13 EpCAM(+)-EV miRNAs compared with healthy individuals. Upon surgical tumour removal, the plasma levels of 8 of these were reduced (miR-16-5p, miR-23a-3p, miR-23b-3p, miR-27a-3p, miR-27b-3p, miR-30b-5p, miR-30c-5p and miR-222-3p). These findings indicate that the miRNAs are of tumour origin and may have potential as non-invasive biomarkers for detection of CRC. This work describes a non-invasive blood-based method for sensitive detection of cancer with potential for clinical use in relation to diagnosis and screening. We used the method to study CRC; however, it is not restricted to this disease. It may in principle be used to study any cancer that release epithelial-derived EVs into circulation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5005366PMC
http://dx.doi.org/10.3402/jev.v5.31488DOI Listing
August 2016

Novel diagnostic and prognostic classifiers for prostate cancer identified by genome-wide microRNA profiling.

Oncotarget 2016 May;7(21):30760-71

Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark.

Purpose: This study investigates the diagnostic and prognostic biomarker potential of miRNAs in prostate cancer (PC).

Results: We identified several new deregulated miRNAs between non-malignant (NM) and PC tissue samples and between more/less aggressive PC subgroups. We also developed and validated a novel 13-miRNA diagnostic classifier with high sensitivity and specificity for PC. Finally, we trained a new 3-miRNA prognostic classifier (miR-185-5p+miR-221-3p+miR-326) that predicted time to biochemical recurrence (BCR) independently of routine clinicopathological variables in a training radical prostatectomy (RP) cohort (n = 126) as well as in two independent validation cohorts (n = 110 and n = 99).

Experimental Design: After RT-qPCR-based profiling of 752 miRNAs in 13 NM and 134 PC tissue samples (cohort 1), we selected 93 top candidate diagnostic/prognostic miRNAs for validation in two independent patient sets (cohort 2: 19 NM and 138 PC; cohort 3: 28 NM and 113 PC samples). Diagnostic potential was assessed by ROC curve analysis and prognostic potential by Kaplan-Meier, uni- and multivariate Cox regression analyses. BCR after RP was used as endpoint.

Conclusions: This is the first report of a miRNA signature with significant independent prognostic value demonstrated in three PC patient cohorts.
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http://dx.doi.org/10.18632/oncotarget.8953DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5058715PMC
May 2016

Evaluation of quantitative miRNA expression platforms in the microRNA quality control (miRQC) study.

Nat Methods 2014 Aug 29;11(8):809-15. Epub 2014 Jun 29.

Center for Medical Genetics, Ghent University, Ghent, Belgium.

MicroRNAs are important negative regulators of protein-coding gene expression and have been studied intensively over the past years. Several measurement platforms have been developed to determine relative miRNA abundance in biological samples using different technologies such as small RNA sequencing, reverse transcription-quantitative PCR (RT-qPCR) and (microarray) hybridization. In this study, we systematically compared 12 commercially available platforms for analysis of microRNA expression. We measured an identical set of 20 standardized positive and negative control samples, including human universal reference RNA, human brain RNA and titrations thereof, human serum samples and synthetic spikes from microRNA family members with varying homology. We developed robust quality metrics to objectively assess platform performance in terms of reproducibility, sensitivity, accuracy, specificity and concordance of differential expression. The results indicate that each method has its strengths and weaknesses, which help to guide informed selection of a quantitative microRNA gene expression platform for particular study goals.
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http://dx.doi.org/10.1038/nmeth.3014DOI Listing
August 2014

microRNAs in nociceptive circuits as predictors of future clinical applications.

Front Mol Neurosci 2013 Oct 17;6:33. Epub 2013 Oct 17.

Department of Physiology and Medical Physics, Division of Physiology, Medical University Innsbruck Innsbruck, Austria.

Neuro-immune alterations in the peripheral and central nervous system play a role in the pathophysiology of chronic pain, and non-coding RNAs - and microRNAs (miRNAs) in particular - regulate both immune and neuronal processes. Specifically, miRNAs control macromolecular complexes in neurons, glia and immune cells and regulate signals used for neuro-immune communication in the pain pathway. Therefore, miRNAs may be hypothesized as critically important master switches modulating chronic pain. In particular, understanding the concerted function of miRNA in the regulation of nociception and endogenous analgesia and defining the importance of miRNAs in the circuitries and cognitive, emotional and behavioral components involved in pain is expected to shed new light on the enigmatic pathophysiology of neuropathic pain, migraine and complex regional pain syndrome. Specific miRNAs may evolve as new druggable molecular targets for pain prevention and relief. Furthermore, predisposing miRNA expression patterns and inter-individual variations and polymorphisms in miRNAs and/or their binding sites may serve as biomarkers for pain and help to predict individual risks for certain types of pain and responsiveness to analgesic drugs. miRNA-based diagnostics are expected to develop into hands-on tools that allow better patient stratification, improved mechanism-based treatment, and targeted prevention strategies for high risk individuals.
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http://dx.doi.org/10.3389/fnmol.2013.00033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3798051PMC
October 2013

Sub-cellular temporal and spatial distribution of electrotransferred LNA/DNA oligomer.

J RNAi Gene Silencing 2013 15;9:479-85. Epub 2013 Mar 15.

Centre National de la Recherche Scientifique, Institut de Pharmacologie et de Biologie Structurale, BP64182, 205 route de Narbonne, F-31077 Toulouse, France ; Université de Toulouse, UPS, IPBS, F-31077 Toulouse, France.

Low biological activity and inefficient targeted delivery in vivo have hindered RNA interference (RNAi)-based therapy from realising its full clinical potential. To overcome these hurdles, progresses have been made to develop new technologies optimizing oligonucleotides chemistry on one hand and achieving its effective delivery on the other hand. In this report, we achieved, by using the electropulsation technique (EP), efficient cellular delivery of chemically-modified oligonucleotide: The locked nucleic acid (LNA)/DNA oligomer. We used single cell level confocal fluorescence microscopy to follow the spatial and temporal distribution of electrotransferred cyanine 5 (Cy5)-labeled LNA/DNA oligomer. We observed that EP allowed LNA/DNA oligomer cellular uptake providing the oligomer a rapid access to the cytoplasm of HeLa cells. Within a few minutes after electrotransfer, Cy5-LNA/DNA oligomers shuttle from cytoplasm to nucleus whereas in absence of pulses application, Cy5-LNA/DNA oligomers were not detected. We then observed a redistribution of the Cy5 fluorescence that accumulated over time into cytoplasmic organelles. To go further and to identify these compartments, we used the HeLa GFP-Rab7 cell line to visualise late endosomes, and lysosomal or mitochondrial specific markers. Our results showed that the EP technique allowed direct entry into the cytoplasm of the Cy5-LNA/DNA oligomer bypassing the endocytosic pathway. However, in absence of pulses application, Cy5-LNA/DNA oligomer were able to enter cells through the endocytosic pathway. We demonstrated that EP is an efficient technique for LNA-based oligonucleotides delivery offering strong advantages by avoiding the endolysosomal compartmentalization, giving a rapid and free access to the cytoplasm and the nucleus where they can find their targets.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3717327PMC
August 2013

Assessing sample and miRNA profile quality in serum and plasma or other biofluids.

Methods 2013 Jan 2;59(1):S1-6. Epub 2012 Oct 2.

Exiqon A/S, Skelstedet 16, 2950 Vedbæk, Denmark.

MicroRNAs (miRNAs) constitute a class of small cellular RNAs (typically 21-23nt) that function as post-transcriptional regulators of gene expression. Current estimates indicate that more than one third of the cellular transcriptome is regulated by miRNAs, although they are relatively few in number (less than 2000 human miRNAs). The high relative stability of miRNA in common clinical tissues and biofluids (e.g. plasma, serum, urine, saliva, etc.) and the ability of miRNA expression profiles to accurately classify discrete tissue types and disease states have positioned miRNA quantification as a promising new tool for a wide range of diagnostic applications. Furthermore miRNAs have been shown to be rapidly released from tissues into the circulation with the development of pathology. To facilitate discovery and clinical development of miRNA-based biomarkers, we developed a genome-wide Locked Nucleic Acid (LNA™)-based miRNA qPCR platform with unparalleled sensitivity and robustness. The platform allows high-throughput profiling of miRNAs from important clinical sources without the need for pre-amplification. Using this system, we have profiled thousands of biofluid samples including blood derived plasma and serum. An extensive quality control (QC) system has been implemented in order to secure technical excellence and reveal any unwanted bias coming from pre-analytical or analytical variables. We present our approaches to sample and RNA QC as well as data QC and normalization. Specifically we have developed normal reference ranges for circulating miRNAs in serum and plasma as well as a hemolysis indicator based on microRNA expression.
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http://dx.doi.org/10.1016/j.ymeth.2012.09.015DOI Listing
January 2013

Profiling microRNAs by real-time PCR.

Methods Mol Biol 2011 ;732:39-54

Exiqon A/S, Vedbaek, Denmark.

A variety of physiological processes are associated with changes in microRNA (miRNA) expression. Analysis of miRNA has been applied to study normal physiology as well as diseased states including cancer. One major challenge in miRNA research is to accurately and practically determine the expression level of miRNAs in various experimental systems. Many genome-wide miRNA expression profiling studies have relied on microarrays technology, and frequently differentially expressed miRNAs have subsequently been confirmed with real-time quantitative PCR studies. Here, we describe how different primer strategies for first-strand cDNA synthesis and PCR amplification can affect measurements of miRNA expression levels. Overcoming the small nature of miRNAs is a difficult task as the short sequence available does not allow for designing primers using standard PCR primer design guidelines. Finally, we demonstrate how to determine differentially expressed miRNAs using a locked nucleic acid-based real-time PCR approach.
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http://dx.doi.org/10.1007/978-1-61779-083-6_4DOI Listing
June 2011

Improved microRNA quantification in total RNA from clinical samples.

Methods 2010 Apr;50(4):S6-9

Exiqon A/S, Skelstedet 16, DK-2950 Vedbaek, Denmark.

microRNAs are small regulatory RNAs that are currently emerging as new biomarkers for cancer and other diseases. In order for biomarkers to be useful in clinical settings, they should be accurately and reliably detected in clinical samples such as formalin fixed paraffin embedded (FFPE) sections and blood serum or plasma. These types of samples represent a challenge in terms of microRNA quantification. A newly developed method for microRNA qPCR using Locked Nucleic Acid (LNA)-enhanced primers enables accurate and reproducible quantification of microRNAs in scarce clinical samples. Here we show that LNA-based microRNA qPCR enables biomarker screening using very low amounts of total RNA from FFPE samples and the results are compared to microarray analysis data. We also present evidence that the addition of a small carrier RNA prior to total RNA extraction, improves microRNA quantification in blood plasma and laser capture microdissected (LCM) sections of FFPE samples.
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http://dx.doi.org/10.1016/j.ymeth.2010.01.006DOI Listing
April 2010

Analysis of separate and combined effects of common variation in KCNJ11 and PPARG on risk of type 2 diabetes.

J Clin Endocrinol Metab 2005 Jun 29;90(6):3629-37. Epub 2005 Mar 29.

Steno Diabetes Center and Hagedorn Research Institute, Niels Steensens Vej 2, DK-2820 Gentofte, Denmark.

The separate and combined effects of the PPARG Pro(12)Ala polymorphism and the KCNJ11 Glu(23)Lys polymorphisms on risk of type 2 diabetes were investigated in relatively large-scale, case-control studies. Separate effects of the variants were examined among 1187/1461 type 2 diabetic patients and 4791/4986 middle-aged, glucose-tolerant subjects. The combined analysis involved 1164 type 2 diabetic patients and 4733 middle-aged, glucose-tolerant subjects. In the separate analyses, the K allele of the KCNJ11 Glu(23)Lys associated with type 2 diabetes (odds ratio, 1.19; P = 0.0002), whereas the PPARG Pro(12)Ala showed no significant association with type 2 diabetes. The combined analysis indicated that the two polymorphisms acted in an additive manner to increase the risk of type 2 diabetes, and we found no evidence for a synergistic interaction between them. Analysis of a model with equal additive effects of the two variants showed that the odds ratio for type 2 diabetes increased with 1.14/risk allele (P = 0.003). Together, the two polymorphisms conferred a population-attributable risk for type 2 diabetes of 28%. In conclusion, our results showed no evidence of a synergistic interaction between the KCNJ11 Glu(23)Lys and PPARG Pro(12)Ala polymorphisms, but indicated that they may act in an additive manner to increase the risk of type 2 diabetes.
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http://dx.doi.org/10.1210/jc.2004-1942DOI Listing
June 2005

PGC-1alpha Gly482Ser polymorphism associates with hypertension among Danish whites.

Hypertension 2005 Apr 28;45(4):565-70. Epub 2005 Feb 28.

Steno Diabetes Center, Niels Steensens Vej 2, NSH2.16, DK-2820 Gentofte, Denmark.

PGC-1alpha is a coactivator of numerous transcription factors and is expressed in tissues with high energy demands and abundant in mitochondria. It is induced in the myocardium on fasting and physical exercise, and cardiac-specific overexpression stimulates mitochondrial biogenesis in mice. The common Gly482Ser polymorphism of PGC-1alpha has previously shown association with arterial hypertension among Austrian men. Thus, we aimed at investigating this relationship in the Danish white population. The Gly482Ser polymorphism was genotyped in a total of 2562 Danish white subjects using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) and a GenoView locked nucleic acid assay (LNA), and the relationships of this variant with blood pressure levels and arterial hypertension were analyzed. Furthermore, we performed a combined analysis of the data from the present study in combination with previously published results. The Ser/Ser genotype was significantly associated with a reduced risk of hypertension and with lower systolic, diastolic, and mean arterial blood pressure levels, predominantly among women. Finally, in a combined analysis using data obtained in both sexes, the Ser/Ser genotype group had an estimated odds ratio of 0.70 (95% confidence interval, 0.56 to 0.86) for hypertension compared with Gly/X carriers (P=0.001). In conclusion, the Ser allele of PGC-1alpha Gly482Ser confers a significantly reduced risk of hypertension in whites. Further studies are needed to elucidate the differential role of this polymorphism in men and women.
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http://dx.doi.org/10.1161/01.HYP.0000158946.53289.24DOI Listing
April 2005

A Blumeria graminis gene family encoding proteins with a C-terminal variable region with homologues in pathogenic fungi.

Gene 2003 Jun;311:181-92

Department of Plant Biology and Biogeochemistry, Risø National Laboratory, P.O. Box 49, DK-4000 Roskilde, Denmark.

In a study aimed at characterising, at the molecular level, the obligate biotrophic fungus Blumeria graminis f. sp. hordei (Bgh), we have identified a novel group of genes, the Egh16H genes, and shown that two of these are up-regulated during primary infection of barley leaves. The genes have partial homology to a previously characterised Bgh gene family, Egh16. Egh16 and Egh16H are subfamilies of a larger multigene family with presently about 15 members identified in Bgh. Egh16H has about ten members, and we show that five of these are expressed as highly conserved mRNAs that are predicted to encode proteins with a C-terminal variable region. Egh16H has high homology to sequences in Magnaporthe grisea and other plant pathogenic fungi, as well as sequences of both the insect pathogen Metarhizium anisopliae and the human pathogen Aspergillus fumigatus. No close homologues of Egh16H were found in the non-pathogenic fungi Neurospora crassa and Aspergillus nidulans. We predict that Egh16H plays a general role in the interaction between pathogenic fungi and their hosts. At present, the large number of gene family members with C-terminal variation appears to be unique for Bgh, and the Egh16/Egh16H gene family is to our knowledge the largest gene family so far characterised in this fungus.
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http://dx.doi.org/10.1016/s0378-1119(03)00610-3DOI Listing
June 2003

Single nucleotide polymorphism genotyping using locked nucleic acid (LNA).

Expert Rev Mol Diagn 2003 Jan;3(1):27-38

Exiqon A/S, Bygstubben 9, DK-2950 Vedbaek, Denmark.

Locked nucleic acid (LNA) is a new class of bicyclic high affinity DNA analogs. LNA-containing oligonucleotides confer significantly increased affinity against their complementary DNA targets, increased mismatch discrimination (delta Tm) and allow full control of the melting point of the hybridization reaction. LNA chemistry is completely compatible with the traditional DNA phosphoramidite chemistry and therefore LNA-DNA mixmer oligonucleotides can be designed with complete freedom for optimal performance. These properties render LNA oligonucleotides very well suited for SNP genotyping and have enabled several approaches for enzyme-independent SNP genotyping based on allele-specific hybridization. In addition, allele-specific PCR assays relying on enzymatically-enhanced discrimination can be improved using LNA-modified oligonucleotides. The use of LNA transforms enzyme-independent genotyping approaches into experimentally simple, robust and cost-effective assays, which are highly suited for genotyping in clinical and industrial settings.
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http://dx.doi.org/10.1586/14737159.3.1.27DOI Listing
January 2003