Karsten Nielsen

Seemingly neutral polymorphic variants may confer immunity to splicing-inactivating mutations: a synonymous SNP in exon 5 of MCAD protects from deleterious mutations in a flanking exonic splicing enhancer.

The idea that point mutations in exons may affect splicing is intriguing and adds an additional layer of complexity when evaluating their possible effects. Even in the best-studied examples, the molecular mechanisms are not fully understood. Here, we use patient cells, model minigenes, and in vitro assays to show that a missense mutation in exon 5 of the medium-chain acyl-CoA dehydrogenase (MCAD) gene primarily causes exon skipping by inactivating a crucial exonic splicing enhancer (ESE), thus leading to loss of a functional protein and to MCAD deficiency. This ESE functions by antagonizing a juxtaposed exonic splicing silencer (ESS) and is necessary to define a suboptimal 3 splice site. Remarkably, a synonymous polymorphic variation in MCAD exon 5 inactivates the ESS, and, although this has no effect on splicing by itself, it makes splicing immune to deleterious mutations in the ESE. Furthermore, the region of MCAD exon 5 that harbors these elements is nearly identical to the exon 7 region of the survival of motor neuron (SMN) genes that contains the deleterious silent mutation in SMN2, indicating a very similar and finely tuned interplay between regulatory elements in these two genes. Our findings illustrate a mechanism for dramatic context-dependent effects of single-nucleotide polymorphisms on gene-expression regulation and show that it is essential that potential deleterious effects of mutations on splicing be evaluated in the context of the relevant haplotype.

Identification and characterization of GFAPκ, a novel glial fibrillary acidic protein isoform

Glial fibrillary acidic protein (GFAP) is the principal component of the intermediary filaments in mature astrocytes of the central nervous system (CNS). The protein consists of three domains: the head, the coiled‐coil, and the tail. Here, we describe the isolation of an evolutionary conserved novel GFAP isoform, GFAPκ, produced by alternative splicing and polyadenylation of the 3′‐region of the human GFAP pre‐mRNA. As a consequence, the resulting human GFAPκ protein harbors a nonconserved C‐terminal tail sequence distinct from the tails of GFAPα, the predominant GFAP isoform, and GFAPε, an isoform which also results from alternative splicing. The head and coiled‐coil rod domains are identical between the three GFAP isoforms. Interestingly, GFAPκ is incapable of forming homomeric filaments, and increasing GFAPκ expression levels causes a collapse of intermediate filaments formed by GFAPα. In searching for a biological relevance of GFAPκ, we noticed that mRNA expression levels of GFAPα, GFAPε, and GFAPκ are gradually increased during development of the embryonic pig brain. However, whereas the GFAPα/GFAPε ratio is constant, the GFAPκ/GFAPε ratio decreases during brain development. Furthermore, in glioblastoma tumors, an increased GFAPκ/GFAPε ratio is detected. Our results suggest that the relative expression level of the GFAPκ isoform could modulate the properties of GFAP intermediate filaments and perhaps thereby influencing the motility of GFAP positive astrocytes and progenitor cells within the CNS.

World Health Organization‐defined classification of myeloproliferative neoplasms: Morphological reproducibility and clinical correlations—The Danish experience

We examined inter‐ and intraobserver reproducibility and concordance between histological diagnosis and independently collected clinical findings in a large series of patients with the major subtypes of myeloproliferative neoplasms (MPNs) and controls. Seven hematopathologists reviewed 272 bone marrow biopsies including 43 controls. Diagnoses were determined according to the 2008 criteria of the World Health Organization (WHO). The participants were blinded to all clinical data except patient age. After initial evaluation all hematopathologists participated in a 3‐day meeting with a leading clinician chaired by an expert hematopathologists. In cases with lack of consensus on fiber grading (nu2009=u200957), a new evaluation was performed. In cases with discordance on morphological diagnosis (nu2009=u2009129), an additional nonblinded evaluation taking clinical data into consideration was carried out. For remaining cases with a lack of concordance between morphological diagnosis and clinical diagnosis (nu2009=u200933), a similar nonblinded evaluation was performed. Consensus on final histological diagnosis and concordance with clinical diagnosis were determined. Blinded histological evaluation resulted in a 53% consensus rate. After re‐evaluation of fiber content, consensus was reached in 60% of cases. Adding clinical data increased the histological consensus to 83%. For cases with a histological consensus, we found a concordance of 71% with the clinicians diagnoses. This is the first study to present a larger cohort of MPN patients mimicking the diagnostic challenges that hematopathologists face in their daily practice. The results support the postulates of the WHO that both morphological and clinical findings are essential for a valid diagnosis Am. J. Hematol. 88:1012–1016, 2013.

A comparison between stereological estimates of mean nuclear volume and DNA flow cytometry in bladder tumours

The correlation between stereological estimate of mean nuclear volume and DNA‐content was studied in 55 human urinary bladder tumours. The DNA‐content was determined by flow cytometry on isolated nuclei stained with ethidium bromide. Trout erythrocytes were used as a biological internal standard providing an accurate determination of the DNA‐histogram. An unbiased estimate of the mean nuclear volume (V̄v) was obtained after standard formaline fixation, paraffin‐embedding, sectioning and hematoxylin‐eosin staining using the equation . Here I0 is the length of an intercept measured in a random direction through a test point, which hit a nucleus. A highly significant correlation was found between V̄v and mean DNA‐content of nuclei (2p = 0.0004). A highly significant correlation was also found between V̄v and the highest DNA‐content present in tumours having cell populations with different DNA content (2p = 0.0016). The mean nuclear volume and the DNA‐content also correlated well with the pathologic grade. Although significant the correlations were far from perfect, which indicates that DNA content and mean nuclear volume may provide partly independent biological information. The methods provide objective, unbiased and reproducible data which may improve the possibility of grading and predicting the disease course of human urinary bladder tumours.

Reelin expression during embryonic development of the pig brain

BackgroundReelin is an extracellular glycoprotein of crucial importance in the developmental organisation of neurons in the mammalian cerebral cortex and other laminated brain regions. The pig possesses a gyrencephalic brain that bears resemblance to the human brain. In order to establish an animal model for neuronal migration disorders in the pig, we have studied the expression pattern and structure of Reelin during pig brain development.ResultsWe determined the sequence of pig Reelin mRNA and protein and identified a high degree of homology to human Reelin. A peak in Reelin mRNA and protein expression is present during the period of major neurogenesis and neuronal migration. This resembles observations for human brain development. Immunohistochemical analysis showed the highest expression of Reelin in the Cajal-Reztius cells of the marginal zone, in resemblance with observations for the developing brain in humans and other mammalian species.ConclusionsWe conclude that the pig might serve as an alternative animal model to study Reelin functions and that manipulation of the pig Reelin could allow the establishment of an animal model for human neuronal migration disorders.

1Identification of genes differentially expressed in the embryonic pig cerebral cortex before and after appearance of gyration

BackgroundMammalian evolution is characterized by a progressive expansion of the surface area of the cerebral cortex, an increase that is accompanied by gyration of the cortical surface. The mechanisms controlling this gyration process are not well characterized but mutational analyses indicate that genes involved in neuronal migration play an important function. Due to the lack of gyration of the rodent brain it is important to establish alternative models to examine brain development during the gyration process. The pig brain is gyrated and accordingly is a candidate alternative model.FindingsIn this study we have identified genes differentially expressed in the pig cerebral cortex before and after appearance of gyration. Pig cortical tissue from two time points in development representing a non-folded, lissencephalic, brain (embryonic day 60) and primary-folded, gyrencephalic, brain (embryonic day 80) were examined by whole genome expression microarray studies. 91 differentially expressed transcripts (fold change >3) were identified. 84 transcripts were annotated and encoding proteins involved in for example neuronal migration, calcium binding, and cytoskeletal structuring. Quantitative real-time PCR was used to confirm the regulation of a subset of the identified genes.ConclusionThis study provides identification of genes which are differentially expressed in the pig cerebral cortex before and after appearance of brain gyration. The identified genes include novel candidate genes which could have functional importance for brain development.

The antagonistic effect of antipsychotic drugs on a HEK293 cell line stably expressing human α1A1-adrenoceptors

Antipsychotic drugs often cause orthostatic hypotension, probably through antagonist action on resistance vessel alpha(1A)-adrenoceptors. Here we have tested this possibility directly using cells transfected with a relevant human alpha(1A)-adrenoceptor splice variant. To determine a splice variant which was relevant, we used quantitative real-time polymerase chain reaction (qPCR) to determine the prevalence in human subcutaneous small arteries of three of the five splice variants ADRA1A_v1-5, which encode functional protein: alpha(1A1)-, alpha(1A3)-, alpha(1A4)-adrenoceptors. Our statistical analysis showed higher transcription levels of alpha(1A1)- than of alpha(1A3)- and alpha(1A4)-adrenoceptors (1.6 and 5.8 times, respectively). We therefore chose to study the alpha(1A1)-adrenoceptor, and the cDNA encoding it was transfected into the Flp-In-293 (modified from HEK-293) cell line to produce a cell line stably expressing a functional form of this splice variant. The expression of recombinant alpha(1A1)-adrenoceptor subtype was confirmed by Western immunoblot analysis, and its functionality demonstrated using a Fura-2 assay by a rise in intracellular calcium concentration ([Ca(2+)](i)) when challenged with phenylephrine (EC(50)=1.61x10(-8) M). From Schild analysis, prazosin, sertindole, risperidone, and haloperidol caused a concentration-dependent, rightward shift of the cumulative concentration-response curves for phenylephrine in cells expressing human recombinant alpha(1A1)-adrenoceptors to yield pK(B) values of 8.40, 8.05, 8.26 and 7.38, respectively. In [7-methoxy-(3)H]-prazosin binding experiments, high expression was seen (B(max)=48.5+/-16.7 pmol/mg protein, +/-S.E.M.) along with high affinity binding to a single site (K(d)=0.210+/-0.034 nM). The pharmacological profiles of recombinant human alpha(1A1)-adrenoceptors in competition binding studies confirmed much higher antagonist affinity of sertindole and risperidone than haloperidol for these receptors. In summary, it can be concluded that there is an approximately 10-fold higher adrenoceptor affinity of risperidone and sertindole for human alpha(1A1)-adrenoceptors compared to haloperidol. These findings are consistent with the observation that risperidone and sertindole have a higher incidence of orthostatic hypotension than haloperidol.

A 7-Gene Signature Depicts the Biochemical Profile of Early Prefibrotic Myelofibrosis

Recent studies have shown that a large proportion of patients classified as essential thrombocythemia (ET) actually have early primary prefibrotic myelofibrosis (prePMF), which implies an inferior prognosis as compared to patients being diagnosed with so-called genuine or true ET. According to the World Health Organization (WHO) 2008 classification, bone marrow histology is a major component in the distinction between these disease entities. However, the differential diagnosis between them may be challenging and several studies have not been able to distinguish between them. Most lately, it has been argued that simple blood tests, including the leukocyte count and plasma lactate dehydrogenase (LDH) may be useful tools to separate genuine ET from prePMF, the latter disease entity more often being featured by anemia, leukocytosis and elevated LDH. Whole blood gene expression profiling was performed in 17 and 9 patients diagnosed with ET and PMF, respectively. Using elevated LDH obtained at the time of diagnosis as a marker of prePMF, a 7-gene signature was identified which correctly predicted the prePMF group with a sensitivity of 100% and a specificity of 89%. The 7 genes included MPO, CEACAM8, CRISP3, MS4A3, CEACAM6, HEMGN, and MMP8, which are genes known to be involved in inflammation, cell adhesion, differentiation and proliferation. Evaluation of bone marrow biopsies and the 7-gene signature showed a concordance rate of 71%, 79%, 62%, and 38%. Our 7-gene signature may be a useful tool to differentiate between genuine ET and prePMF but needs to be validated in a larger cohort of “ET” patients.

Abstract B32: High-throughput solution for microRNA (miRNA) isolation from stabilized whole blood in a GLP setting: Development and comparison to established procedures.

Introduction: Beside the analysis of messenger RNA (mRNA) expression changes, profiling of miRNAs in human whole blood holds much promise for the development of genetic markers of disease. Many scientific groups in academia and in life science companies investigate the use of miRNAs as diagnostic tools at the moment. As these small RNAs become more and more important, effective and reliable isolation procedures are urgently needed. Especially standardization and exclusion of human errors require new automated solutions which are able to replace established manual processes without losing quality and quantity of the needed analytes. In this study we compare a newly developed and optimized procedure on QIAGEN9s BioRobot platform to previously published automated (Kruhoffer et al., 2007, JMD 9: 452–458) as well as manual procedures. Material & Methods: Whole blood from consented healthy adults was collected into PAXgene Blood RNA Tubes and subjected to RNA isolation. The RNA quality and quantity were determined by using a Beckman photometer and Agilent Bioanalyzer. miRNA yields were determined by quantitative RT-PCR using commercially available probe based (Life Technologies) assays on a BioMark (Fluidigm) instrument. Due to the small reaction volume of 9 nanoliters on the BioMark arrays a pre-amplification of the cDNA was performed. Therefore 14 amplification cycles were run with a 1:100 diluted primer set which was the same that was used in the following detection assays. Results: Compared to the originally published partly automated method the workflow could be further streamlined resulting in a fully automated procedure. With this automation the time needed for manual steps could be reduced by more than 50%. In parallel, the quality and quantity of the isolated RNA (large and small RNA species) was not affected. This is demonstrated by high RIN values (>7) and low DNA contamination levels Conclusion: This comparison demonstrates the high efficiency of this optimized, fully automated isolation procedure for miRNAs from stabilized whole blood. The new procedure for purification of miRNAs provides the standardization required in a GLP setting. All miRNA enrichment protocols, the PAXgene Blood miRNA Kit and the PAXgene Blood RNA MDx Kit are for research use only. Not for use in diagnostic procedures. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B32.