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  • 1.
    Alagaratnam, S.
    et al.
    DNV GL, Grp Technol & Res, Precis Med Programme, Hovik, Norway..
    Pedersen, G. Meldre
    DNV GL, Grp Technol & Res, Precis Med Programme, Hovik, Norway..
    McAdam, S.
    DNV GL, Digital Hlth Incubator, Digital Solut, Hovik, Norway..
    Wirta, Valtteri
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi. Karolinska Inst, Dept Microbiol Tumor & Cell Biol, Sci Life Lab, Stockholm, Sweden..
    Lundeberg, Joakim
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi.
    Duno, M.
    Copenhagen Univ Hosp, Dept Clin Genet, Copenhagen, Denmark..
    Wadt, K. A. W.
    Copenhagen Univ Hosp, Dept Clin Genet, Copenhagen, Denmark..
    Rossing, M.
    Copenhagen Univ Hosp, Ctr Genom Med, Copenhagen, Denmark..
    Jonsson, J. J.
    Univ Iceland, Dept Genet & Mol Med, Landspitali Natl Univ Hosp, Reykjavik, Iceland.;Univ Iceland, Dept Biochem & Mol Biol, Fac Med, Reykjavik, Iceland..
    Saarela, J.
    Ctr Mol Med Norway, Oslo, Norway.;Univ Helsinki, Inst Mol Med Finland, Helsinki, Finland..
    Undlien, D.
    Oslo Univ Hosp, Dept Med Genet, Oslo, Norway..
    Quality improvement in clinical NGS through a peer-driven Nordic collaboration2019Ingår i: European Journal of Human Genetics, ISSN 1018-4813, E-ISSN 1476-5438, Vol. 27, s. 1622-1623Artikel i tidskrift (Övrigt vetenskapligt)
  • 2.
    Eisfeldt, Jesper
    et al.
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden..
    Lundin, Johanna
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden..
    Pettersson, Maria
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden..
    Kvarnung, Malin
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden..
    Lieden, Agne
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden..
    Sahlin, Ellika
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden..
    Lagerstedt, Kristina
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden..
    Martin, Marcel
    Nbis, Stockholm, Sweden..
    Ygberg, Sofia
    Karolinska Inst, Inst Womens & Childrens Hlth, Neuropediat Unit, Stockholm, Sweden..
    Bjerin, Olof
    Karolinska Inst, Inst Womens & Childrens Hlth, Neuropediat Unit, Stockholm, Sweden..
    Stranneheim, Henrik
    Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden..
    Wedell, Anna
    Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden..
    Nordenskjold, Magnus
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden..
    Soller, Maria Johansson
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden..
    Nordgren, Ann
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden..
    Wirta, Valtteri
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Nilsson, Daniel
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden..
    Lindstrand, Anna
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden..
    From cytogenetics to cytogenomics whole genome sequencing as a comprehensive genetic test in rare disease diagnostics2019Ingår i: Molecular Cytogenetics, ISSN 1755-8166, E-ISSN 1755-8166, Vol. 12Artikel i tidskrift (Övrigt vetenskapligt)
  • 3. Eisfeldt, Jesper
    et al.
    Nazaryan-Petersen, Lusine
    Lundin, Johanna Lundin
    Pettersson, Maria
    Nilsson, Daniel
    Wincent, Josephine
    Lieden, Agne
    Vezzi, Francesco
    Wirta, Valteri
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Käller, Max
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Duelund, Tina
    Houssari, Rayan
    Pignata, Laura
    Bak, Mads
    Tommerup, Niels
    Lundberg, Elisabeth Syk
    Tumer, Zeynep
    Lindstrand, Anna
    Whole genome characterization of array defined clustered CNVs reveals two distinct complex rearrangement subclasses generated through either non homologous repair or template switching2017Ingår i: Molecular Cytogenetics, ISSN 1755-8166, E-ISSN 1755-8166, Vol. 10Artikel i tidskrift (Övrigt vetenskapligt)
  • 4. Hilson, P.
    et al.
    Allemeersch, J.
    Altmann, T.
    Aubourg, S.
    Avon, A.
    Beynon, J.
    Bhalerao, R. P.
    Bitton, F.
    Caboche, M.
    Cannoot, B.
    Chardakov, V.
    Cognet-Holliger, C.
    Colot, V.
    Crowe, M.
    Darimont, C.
    Durinck, S.
    Eickhoff, H.
    de Longevialle, A. F.
    Farmer, E. E.
    Grant, M.
    Kuiper, M. T. R.
    Lehrach, H.
    Leon, C.
    Leyva, A.
    Lundeberg, Joakim
    KTH, Tidigare Institutioner, Bioteknologi.
    Lurin, C.
    Moreau, Y.
    Nietfeld, W.
    Paz-Ares, J.
    Reymond, P.
    Rouze, P.
    Sandberg, G.
    Segura, M. D.
    Serizet, C.
    Tabrett, A.
    Taconnat, L.
    Thareau, V.
    Van Hummelen, P.
    Vercruysse, S.
    Vuylsteke, M.
    Weingartner, M.
    Weisbeek, P. J.
    Wirta, Valtteri
    KTH, Tidigare Institutioner, Bioteknologi.
    Wittink, F. R. A.
    Zabeau, M.
    Small, I.
    Versatile gene-specific sequence tags for Arabidopsis functional genomics: Trancript profiling and reverse genetics applications2004Ingår i: Genome Research, ISSN 1088-9051, E-ISSN 1549-5469, Vol. 14, nr 10B, s. 2176-2189Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Microarray transcript profiling and RNA interference are two new technologies crucial for large-scale gene function studies in multicellular eukaryotes. Both rely on sequence-specific hybridization between complementary nucleic acid strands, inciting us to create a collection of gene-specific sequence tags (GSTs) representing at least 21,500 Arabidopsis genes and which are compatible with both approaches. The GSTs were carefully selected to ensure that each of them shared no significant similarity with any other region in the Arabidopsis genome. They were synthesized by PCR amplification from genomic DNA. Spotted microarrays fabricated from the GSTs show good dynamic range, specificity, and sensitivity in transcript profiling experiments. The GSTs have also been transferred to bacterial plasmid vectors via recombinational cloning protocols. These cloned GSTs constitute the ideal starting point for a variety of functional approaches, including reverse genetics. We have subcloned GSTs on a large scale into vectors designed for gene silencing in plant cells. We show that in planta expression of GST hairpin RNA results in the expected phenotypes in silenced Arabidopsis lines. These versatile GST resources provide novel and powerful tools for functional genomics.

  • 5. Hofmeister, Wolfgang
    et al.
    Nilsson, Daniel
    Topa, Alexandra
    Anderlid, Britt-Marie
    Darki, Fahimeh
    Matsson, Hans
    Paez, Isabel Tapia
    Klingberg, Torkel
    Samuelsson, Lena
    Wirta, Valtteri
    KTH, Skolan för bioteknologi (BIO), Genteknologi.
    Vezzi, Francesco
    Kere, Juha
    Nordenskjold, Magnus
    Lundberg, Elisabeth Syk
    Lindstrand, Anna
    CTNND2: a candidate gene for reading problems and mild intellectual disability2015Ingår i: J MED GENET, ISSN 0022-2593, Vol. 52, nr 2, s. 111-122Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background Cytogenetically visible chromosomal translocations are highly informative as they can pinpoint strong effect genes even in complex genetic disorders. Methods and results Here, we report a mother and daughter, both with borderline intelligence and learning problems within the dyslexia spectrum, and two apparently balanced reciprocal translocations: t(1;8)(p22; q24) and t(5; 18)(p15; q11). By low coverage mate-pair whole-genome sequencing, we were able to pinpoint the genomic breakpoints to 2 kb intervals. By direct sequencing, we then located the chromosome 5p breakpoint to intron 9 of CTNND2. An additional case with a 163 kb microdeletion exclusively involving CTNND2 was identified with genome-wide array comparative genomic hybridisation. This microdeletion at 5p15.2 is also present in mosaic state in the patient's mother but absent from the healthy siblings. We then investigated the effect of CTNND2 polymorphisms on normal variability and identified a polymorphism (rs2561622) with significant effect on phonological ability and white matter volume in the left frontal lobe, close to cortical regions previously associated with phonological processing. Finally, given the potential role of CTNND2 in neuron motility, we used morpholino knockdown in zebrafish embryos to assess its effects on neuronal migration in vivo. Analysis of the zebrafish forebrain revealed a subpopulation of neurons misplaced between the diencephalon and telencephalon. Conclusions Taken together, our human genetic and in vivo data suggest that defective migration of subpopulations of neuronal cells due to haploinsufficiency of CTNND2 contribute to the cognitive dysfunction in our patients.

  • 6.
    Laurell, Cecilia
    et al.
    KTH, Skolan för bioteknologi (BIO), Genteknologi.
    Wirta, Valtteri
    KTH, Skolan för bioteknologi (BIO), Genteknologi.
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Genteknologi.
    Lundeberg, Joakim
    KTH, Skolan för bioteknologi (BIO), Genteknologi.
    Comparative analysis of a 3' end tag PCR and a linear RNA amplification approach for microarray analysis2007Ingår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 127, nr 4, s. 638-646Artikel i tidskrift (Refereegranskat)
    Abstract [en]

     Background: Various types of amplification techniques have been developed in order to enable microarray gene expression analysis when the amount of starting material is limited. The two main strategies are linear amplification, using in vitro transcription, and exponential amplification, based on PCR. We have evaluated the performance of a linear and an in-house developed exponential amplification protocol that relies on 3' end tag sequences. We used 100 ng total RNA as starting material for amplification and compared the results with data from hybridizations with unamplified mRNA and total RNA.

    Results: Preservation of expression ratios after amplification was examined comparing 1092 ratios obtained with amplification protocols to those obtained with standard labelling of mRNA. The Pearson correlations were 0.61 and 0.84, respectively, for the two linear amplification replicates and 0.76 and 0.80 for the two exponential amplification replicates. The correlations between repeated amplifications was 0.82 with the exponential method and 0.63 with the linear, indicating a better reproducibility with the PCR-based approach.

    Conclusion: Both amplification methods generated results in agreement with unamplified material. In this study, the PCR-based method was more reproducible than in vitro transcription amplification. Advantages with the in-house developed method are the lower cost since it is non-commercial and that the PCR generated product offers compatibility with both sense and antisense arrays.

  • 7.
    Lindstrand, Anna
    et al.
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden.;Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden..
    Eisfeldt, Jesper
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden.;Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden.;Karolinska Inst, Sci Life Lab, Stockholm, Sweden..
    Pettersson, Maria
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden.;Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden..
    Carvalho, Claudia M. B.
    Baylor Coll Med, Dept Mol & Human Genet, Houston, TX 77030 USA..
    Kvarnung, Malin
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden.;Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden..
    Grigelioniene, Giedre
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden.;Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden..
    Anderlid, Britt-Marie
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden.;Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden..
    Bjerin, Olof
    Karolinska Inst, Dept Womens & Childrens Hlth, Stockholm, Sweden..
    Gustavsson, Peter
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden.;Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden..
    Hammarsjö, Anna
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden.;Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden..
    Georgii-Hemming, Patrik
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden..
    Iwarsson, Erik
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden.;Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden..
    Johansson-Soller, Maria
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden.;Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden..
    Lagerstedt-Robinson, Kristina
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden.;Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden..
    Lieden, Agne
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden.;Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden..
    Magnusson, Mans
    Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden.;Karolinska Inst, Sci Life Lab, Stockholm, Sweden.;Karolinska Univ Hosp, Ctr Inherited Metab Dis, Stockholm, Sweden..
    Martin, Marcel
    Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, Natl Bioinformat Infrastruct Sweden, Stockholm, Sweden..
    Malmgren, Helena
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden.;Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden..
    Nordenskjöld, Magnus
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden.;Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden..
    Norling, Ameli
    Karolinska Inst, Dept Womens & Childrens Hlth, Stockholm, Sweden..
    Sahlin, Ellika
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden.;Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden..
    Stranneheim, Henrik
    Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden.;Karolinska Univ Hosp, Ctr Inherited Metab Dis, Stockholm, Sweden..
    Tham, Emma
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden.;Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden..
    Wincent, Josephine
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden.;Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden..
    Ygberg, Sofia
    Karolinska Univ Hosp, Ctr Inherited Metab Dis, Stockholm, Sweden..
    Wedell, Anna
    Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden.;Karolinska Univ Hosp, Ctr Inherited Metab Dis, Stockholm, Sweden..
    Wirta, Valtteri
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi.
    Nordgren, Ann
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden.;Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden..
    Lundin, Johanna
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden.;Baylor Coll Med, Dept Mol & Human Genet, Houston, TX 77030 USA..
    Nilsson, Daniel
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden.;Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden.;Karolinska Inst, Sci Life Lab, Stockholm, Sweden..
    From cytogenetics to cytogenomics: whole-genome sequencing as a first-line test comprehensively captures the diverse spectrum of disease-causing genetic variation underlying intellectual disability2019Ingår i: Genome Medicine, ISSN 1756-994X, E-ISSN 1756-994X, Vol. 11, nr 1, artikel-id 68Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    BackgroundSince different types of genetic variants, from single nucleotide variants (SNVs) to large chromosomal rearrangements, underlie intellectual disability, we evaluated the use of whole-genome sequencing (WGS) rather than chromosomal microarray analysis (CMA) as a first-line genetic diagnostic test.MethodsWe analyzed three cohorts with short-read WGS: (i) a retrospective cohort with validated copy number variants (CNVs) (cohort 1, n=68), (ii) individuals referred for monogenic multi-gene panels (cohort 2, n=156), and (iii) 100 prospective, consecutive cases referred to our center for CMA (cohort 3). Bioinformatic tools developed include FindSV, SVDB, Rhocall, Rhoviz, and vcf2cytosure.ResultsFirst, we validated our structural variant (SV)-calling pipeline on cohort 1, consisting of three trisomies and 79 deletions and duplications with a median size of 850kb (min 500bp, max 155Mb). All variants were detected. Second, we utilized the same pipeline in cohort 2 and analyzed with monogenic WGS panels, increasing the diagnostic yield to 8%. Next, cohort 3 was analyzed by both CMA and WGS. The WGS data was processed for large (>10kb) SVs genome-wide and for exonic SVs and SNVs in a panel of 887 genes linked to intellectual disability as well as genes matched to patient-specific Human Phenotype Ontology (HPO) phenotypes. This yielded a total of 25 pathogenic variants (SNVs or SVs), of which 12 were detected by CMA as well. We also applied short tandem repeat (STR) expansion detection and discovered one pathologic expansion in ATXN7. Finally, a case of Prader-Willi syndrome with uniparental disomy (UPD) was validated in the WGS data.Important positional information was obtained in all cohorts. Remarkably, 7% of the analyzed cases harbored complex structural variants, as exemplified by a ring chromosome and two duplications found to be an insertional translocation and part of a cryptic unbalanced translocation, respectively.ConclusionThe overall diagnostic rate of 27% was more than doubled compared to clinical microarray (12%). Using WGS, we detected a wide range of SVs with high accuracy. Since the WGS data also allowed for analysis of SNVs, UPD, and STRs, it represents a powerful comprehensive genetic test in a clinical diagnostic laboratory setting.

  • 8.
    Lundin, Karin E.
    et al.
    Karolinska Inst, Dept Lab Med, Clin Res Ctr, Novum, SE-14186 Stockholm, Sweden..
    Wang, Qing
    Karolinska Inst, Dept Lab Med, Clin Res Ctr, Novum, SE-14186 Stockholm, Sweden..
    Hamasy, Abdulrahman
    Karolinska Inst, Dept Lab Med, Clin Res Ctr, Novum, SE-14186 Stockholm, Sweden.;Hawler Med Univ, Coll Pharm, Dept Clin Anal, Erbil, Kurdistan Regio, Iraq..
    Marits, Per
    Karolinska Univ Hosp, Dept Clin Immunol, SE-14186 Stockholm, Sweden..
    Uzunel, Mehmet
    Karolinska Univ Hosp, Dept Clin Immunol, SE-14186 Stockholm, Sweden..
    Wirta, Valtteri
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Wikstrom, Ann-Charlotte
    Karolinska Univ Hosp, Dept Clin Immunol, SE-14186 Stockholm, Sweden..
    Fasth, Anders
    Univ Gothenburg, Sahlgrenska Acad, Inst Clin Sci, Dept Pediat, SE-41685 Gothenburg, Sweden..
    Ekwall, Olov
    Univ Gothenburg, Sahlgrenska Acad, Inst Clin Sci, Dept Pediat, SE-41685 Gothenburg, Sweden.;Univ Gothenburg, Sahlgrenska Acad, Inst Med, Dept Rheumatol & Inflammat Res, SE-41685 Gothenburg, Sweden..
    Smith, C. I. Edvard
    Karolinska Inst, Dept Lab Med, Clin Res Ctr, Novum, SE-14186 Stockholm, Sweden..
    Eleven percent intact PGM3 in a severely immunodeficient patient with a novel splice-site mutation, a case report2018Ingår i: BMC Pediatrics, ISSN 1471-2431, E-ISSN 1471-2431, Vol. 18, artikel-id 285Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: A novel immunodeficiency, frequently accompanied by high serum-IgE, and caused by mutations in the PGM3 gene was described in 2014. To date there are no unique phenotype characteristics for PGM3 deficiency. PGM3 encodes a carbohydrate-modifying enzyme, phosphoglucomutase 3. Null-mutations are quite likely lethal, and to date only missense mutations or small deletions have been reported. Such mutations frequently cause a combination of reduced enzyme activity and protein instability, complicating determination of the enzyme level needed for survival. Here we present the first patient with a homozygous splice-modifying mutation in the PGM3 gene. An A > G substitution at position c.871 +3 (transcript NM_001199917) is causing a deletion of exon 7 in the majority of PGM3 transcripts. In addition, this case further increases the clinical phenotypes of immunodeficiency caused by PGM3 mutations. Case presentation: We describe the symptoms of a 3-year-old girl who was severely growth retarded, had vascular malformations, extensive eczema, multiple food-allergies, and was prone to infections. Unlike the majority of reported PGM3 deficient patients she lacked skeletal dysplasia and had normal neurocognitive development. In addition to the high serum-IgE, she displayed altered T cell numbers with reduced naive CD4(+) and CD8(+) T-cells, increased number of activated effector memory CD8(+) T cells and aberrant T-cell functions. The patient was homozygous for a new hypomorphic, splice-modifying mutation in the PGM3 gene, causing severely reduced mRNA levels. In the patient's cells, we observed 5% intact mRNA and approximately 11% of the protein levels seen in healthy controls. Treatment with allogeneic hematopoietic stem cell therapy was planned, but unfortunately the clinical condition deteriorated with multi-organ failure, which led to her death at 3 years of age. Conclusions: There is still no specific phenotype identified that distinguishes immunodeficiency caused by PGM3 mutations from other forms of immunodeficiency. The patient described here yields new information on the phenotypic variability among these patients. In addition, since all the synthesized protein is wild-type, it is possible for the first time to estimate the enzyme activity in vivo. The results suggest that1/10 of the normal PGM3 level is sufficient for survival but that it is insufficient for accurate carbohydrate processing.

  • 9. Meletis, Konstantinos
    et al.
    Wirta, Valtteri
    KTH, Skolan för bioteknologi (BIO).
    Hede, Sanna-Maria
    Nistér, Monica
    Lundeberg, Joakim
    KTH, Skolan för bioteknologi (BIO).
    Frisén, Jonas
    p53 suppresses the self-renewal of adult neural stem cells2006Ingår i: Development, ISSN 0950-1991, E-ISSN 1477-9129, Vol. 133, nr 2, s. 363-369Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    There is increasing evidence that tumors are heterogeneous and that a subset of cells act as cancer stem cells. Several proto-oncogenes and tumor suppressors control key aspects of stem cell function, suggesting that similar mechanisms control normal and cancer stem cell properties. We show here that the prototypical tumor suppressor p53, which plays an important role in brain tumor initiation and growth, is expressed in the neural stem cell lineage in the adult brain. p53 negatively regulates proliferation and survival, and thereby self-renewal, of neural stem cells. Analysis of the neural stem cell transcriptome identified the dysregulation of several cell cycle regulators in the absence of p53, most notably a pronounced downregulation of p21 expression. These data implicate p53 as a suppressor of tissue and cancer stem cell self-renewal.

  • 10.
    Nazaryan-Petersen, L.
    et al.
    Univ Copenhagen, Inst Cellular & Mol Med, Wilhelm Johannsen Ctr Funct Genome Res, Copenhagen, Denmark..
    Eisfeldt, J.
    Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden.;Karolinska Inst Sci Pk, Sci Life Lab, Solna, Sweden..
    Lundin, J.
    Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden.;Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden..
    Pettersson, M.
    Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden..
    Nilsson, D.
    Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden.;Karolinska Inst Sci Pk, Sci Life Lab, Solna, Sweden.;Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden..
    Wincent, J.
    Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden..
    Lieden, A.
    Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden.;Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden..
    Vezzi, F.
    Stockholm Univ, Dept Biochem & Biophys, SciLifeLab, Stockholm, Sweden..
    Wirta, Valtteri
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Käller, Max
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Duelund, T.
    Rigshosp, Copenhagen Univ Hosp, Dept Clin Genet, Kennedy Ctr, Copenhagen, Denmark..
    Houssari, R.
    Rigshosp, Copenhagen Univ Hosp, Dept Clin Genet, Kennedy Ctr, Copenhagen, Denmark..
    Pignata, L.
    Rigshosp, Copenhagen Univ Hosp, Dept Clin Genet, Kennedy Ctr, Copenhagen, Denmark..
    Bak, M.
    Univ Copenhagen, Inst Cellular & Mol Med, Wilhelm Johannsen Ctr Funct Genome Res, Copenhagen, Denmark..
    Tommerup, N.
    Univ Copenhagen, Inst Cellular & Mol Med, Wilhelm Johannsen Ctr Funct Genome Res, Copenhagen, Denmark..
    Lundberg, E. S.
    Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden.;Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden..
    Tumer, Z.
    Rigshosp, Copenhagen Univ Hosp, Dept Clin Genet, Kennedy Ctr, Copenhagen, Denmark..
    Lindstrand, A.
    Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden.;Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden..
    Whole genome characterization of array defined clustered CNVs reveals two distinct complex rearrangement subclasses generated through either non-homologous repair or template switching2018Ingår i: European Journal of Human Genetics, ISSN 1018-4813, E-ISSN 1476-5438, Vol. 26, s. 60-60Artikel i tidskrift (Övrigt vetenskapligt)
  • 11.
    Nazaryan-Petersen, Lusine
    et al.
    Univ Copenhagen, Wilhelm Johannsen Ctr Funct Genome Res, Inst Cellular & Mol Med, Copenhagen, Denmark..
    Eisfeldt, Jesper
    Karolinska Inst, Dept Mol Med & Surg, Ctr Mol Med, Stockholm, Sweden.;Karolinska Inst Sci Pk, Sci Life Lab, Solna, Sweden..
    Pettersson, Maria
    Karolinska Inst, Dept Mol Med & Surg, Ctr Mol Med, Stockholm, Sweden..
    Lundin, Johanna
    Karolinska Inst, Dept Mol Med & Surg, Ctr Mol Med, Stockholm, Sweden.;Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden..
    Nilsson, Daniel
    Karolinska Inst, Dept Mol Med & Surg, Ctr Mol Med, Stockholm, Sweden.;Karolinska Inst Sci Pk, Sci Life Lab, Solna, Sweden.;Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden..
    Wincent, Josephine
    Karolinska Inst, Dept Mol Med & Surg, Ctr Mol Med, Stockholm, Sweden.;Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden..
    Lieden, Agne
    Karolinska Inst, Dept Mol Med & Surg, Ctr Mol Med, Stockholm, Sweden.;Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden..
    Lovmar, Lovisa
    Sahlgrens Univ Hosp, Dept Clin Genet, Gothenburg, Sweden..
    Ottosson, Jesper
    Sahlgrens Univ Hosp, Dept Clin Genet, Gothenburg, Sweden..
    Gacic, Jelena
    Linkoping Univ Hosp, Dept Clin Genet, Linkoping, Sweden..
    Makitie, Outi
    Karolinska Inst, Dept Mol Med & Surg, Ctr Mol Med, Stockholm, Sweden.;Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden.;Univ Helsinki, Childrens Hosp, Helsinki, Finland.;Helsinki Univ Hosp, Helsinki, Finland.;Folkhalsan Inst Genet, Helsinki, Finland..
    Nordgren, Ann
    Karolinska Inst, Dept Mol Med & Surg, Ctr Mol Med, Stockholm, Sweden.;Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden..
    Vezzi, Francesco
    Stockholm Univ, SciLifeLab, Dept Biochem & Biophys, Stockholm, Sweden.;Devyser AB, Instrumentvagen 19, Hagersten, Sweden..
    Wirta, Valtteri
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH). KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Käller, Max
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH). KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Hjortshoj, Tina Duelund
    Rigshosp, Kennedy Ctr, Dept Clin Genet, Copenhagen Univ Hosp, Glostrup, Denmark..
    Jespersgaard, Cathrine
    Rigshosp, Kennedy Ctr, Dept Clin Genet, Copenhagen Univ Hosp, Glostrup, Denmark..
    Houssari, Rayan
    Rigshosp, Kennedy Ctr, Dept Clin Genet, Copenhagen Univ Hosp, Glostrup, Denmark..
    Pignata, Laura
    Rigshosp, Kennedy Ctr, Dept Clin Genet, Copenhagen Univ Hosp, Glostrup, Denmark..
    Bak, Mads
    Univ Copenhagen, Wilhelm Johannsen Ctr Funct Genome Res, Inst Cellular & Mol Med, Copenhagen, Denmark..
    Tommerup, Niels
    Univ Copenhagen, Wilhelm Johannsen Ctr Funct Genome Res, Inst Cellular & Mol Med, Copenhagen, Denmark..
    Lundberg, Elisabeth Syk
    Karolinska Inst, Dept Mol Med & Surg, Ctr Mol Med, Stockholm, Sweden.;Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden..
    Tumer, Zeynep
    Rigshosp, Kennedy Ctr, Dept Clin Genet, Copenhagen Univ Hosp, Glostrup, Denmark.;Univ Copenhagen, Fac Hlth & Med Sci, Dept Clin Med, Copenhagen, Denmark..
    Lindstrand, Anna
    Karolinska Inst, Dept Mol Med & Surg, Ctr Mol Med, Stockholm, Sweden.;Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden..
    Replicative and non-replicative mechanisms in the formation of clustered CNVs are indicated by whole genome characterization2018Ingår i: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 14, nr 11, artikel-id e1007780Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Clustered copy number variants (CNVs) as detected by chromosomal microarray analysis (CMA) are often reported as germline chromothripsis. However, such cases might need further investigations by massive parallel whole genome sequencing (WGS) in order to accurately define the underlying complex rearrangement, predict the occurrence mechanisms and identify additional complexities. Here, we utilized WGS to delineate the rearrangement structure of 21 clustered CNV carriers first investigated by CMA and identified a total of 83 breakpoint junctions (BPJs). The rearrangements were further sub-classified depending on the patterns observed: I) Cases with only deletions (n = 8) often had additional structural rearrangements, such as insertions and inversions typical to chromothripsis; II) cases with only duplications (n = 7) or III) combinations of deletions and duplications (n = 6) demonstrated mostly interspersed duplications and BPJs enriched with microhomology. In two cases the rearrangement mutational signatures indicated both a breakage-fusion-bridge cycle process and haltered formation of a ring chromosome. Finally, we observed two cases with Alu- and LINE-mediated rearrangements as well as two unrelated individuals with seemingly identical clustered CNVs on 2p25.3, possibly a rare European founder rearrangement. In conclusion, through detailed characterization of the derivative chromosomes we show that multiple mechanisms are likely involved in the formation of clustered CNVs and add further evidence for chromoanagenesis mechanisms in both "simple" and highly complex chromosomal rearrangements. Finally, WGS characterization adds positional information, important for a correct clinical interpretation and deciphering mechanisms involved in the formation of these rearrangements.

  • 12.
    Nilsson, D.
    et al.
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden.;Karolinska Inst, Dept Mol Med & Surg, SciLifeLab, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden..
    Eisfeldt, J.
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden.;Karolinska Inst, Dept Mol Med & Surg, SciLifeLab, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden..
    Lundin, J.
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden.;Karolinska Inst, Dept Womens & Childrens Hlth, Stockholm, Sweden..
    Pettersson, M.
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden.;Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden..
    Kvarnung, M.
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden.;Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden..
    Lieden, A.
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden.;Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden..
    Sahlin, E.
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden.;Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden..
    Lagerstedt, K.
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden.;Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden..
    Martin, M.
    Stockholm Univ, Natl Bioinformat Infrastruct Sweden, Sci Life Lab, Dept Biochem & Biophys, Solna, Sweden..
    Ygberg, S.
    Karolinska Inst, Inst Womens & Childrens Hlth, Neuropediat Unit, Stockholm, Sweden.;Karolinska Univ Hosp, Ctr Inherited Metab Dis, Stockholm, Sweden..
    Bjerin, O.
    Karolinska Inst, Inst Womens & Childrens Hlth, Neuropediat Unit, Stockholm, Sweden..
    Stranneheim, H.
    Karolinska Inst, Dept Mol Med & Surg, SciLifeLab, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden.;Karolinska Univ Hosp, Ctr Inherited Metab Dis, Stockholm, Sweden..
    Wedell, A.
    Karolinska Inst, Dept Mol Med & Surg, SciLifeLab, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden.;Karolinska Univ Hosp, Ctr Inherited Metab Dis, Stockholm, Sweden..
    Nordenskjold, M.
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden.;Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden..
    Soller, M. Johansson
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden.;Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden..
    Nordgren, A.
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden.;Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden..
    Wirta, Valtteri
    KTH, Skolan för bioteknologi (BIO), Centra, KTH Genome Center. KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi. Karolinska Inst, Dept Microbiol Tumor & Cell Biol, SciLifeLab, Stockholm, Sweden..
    Lindstrand, A.
    Karolinska Univ Hosp, Dept Clin Genet, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden.;Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden..
    From cytogenetics to cytogenomics: whole genome sequencing as a comprehensive genetic test in rare disease diagnostics2019Ingår i: European Journal of Human Genetics, ISSN 1018-4813, E-ISSN 1476-5438, Vol. 27, s. 1666-1667Artikel i tidskrift (Övrigt vetenskapligt)
    Abstract [en]

    Rare genetic diseases are caused by different types of genetic variants, from single nucleotide variants (SNVs) to large chromosomal rearrangements. Recent data indicates that whole genome sequencing (WGS) may be used as a comprehensive test to identify multiple types of pathologic genetic aberrations in a single analysis.

    We present FindSV, a bioinformatic pipeline for detection of balanced (inversions and translocations) and unbalanced (deletions and duplications) structural variants (SVs). First, FindSV was tested on 106 validated deletions and duplications with a median size of 850 kb (min: 511 bp, max: 155 Mb). All variants were detected. Second, we demonstrated the clinical utility in 138 monogenic WGS panels. SV analysis yielded 11 diagnostic findings (8%). Remarkably, a complex structural rearrangement involving two clustered deletions disrupting SCN1A, SCN2A, and SCN3A was identified in a three months old girl with epileptic encephalopathy. Finally, 100 consecutive samples referred for clinical microarray were also analyzed by WGS. The WGS data was screened for large (>2 kbp) SVs genome wide, processed for visualization in our clinical routine arrayCGH workflow with the newly developed tool vcf2cytosure, and for exonic SVs and SNVs in a panel of 700 genes linked to intellectual disability. We also applied short tandem repeat (STR) expansion detection and discovered one pathologic expansion in ATXN7. The diagnostic rate (29%) was doubled compared to clinical microarray (12%).

    In conclusion, using WGS we have detected a wide range of structural variation with high accuracy, confirming it a powerful comprehensive genetic test in a clinical diagnostic laboratory setting.

  • 13. Nilsson, D.
    et al.
    Pettersson, M.
    Gustavsson, P.
    Förster, A.
    Hofmeister, W.
    Wincent, J.
    Zachariadis, V.
    Anderlid, B. -M
    Nordgren, A.
    Mäkitie, O.
    Wirta, Valtteri
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Käller, Max
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Vezzi, F.
    Lupski, J. R.
    Nordenskjöld, M.
    Syk Lundberg, E.
    Carvalho, C. M. B.
    Lindstrand, A.
    Whole-Genome Sequencing of Cytogenetically Balanced Chromosome Translocations Identifies Potentially Pathological Gene Disruptions and Highlights the Importance of Microhomology in the Mechanism of Formation2017Ingår i: Human Mutation, ISSN 1059-7794, E-ISSN 1098-1004, Vol. 38, nr 2, s. 180-192Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Most balanced translocations are thought to result mechanistically from nonhomologous end joining or, in rare cases of recurrent events, by nonallelic homologous recombination. Here, we use low-coverage mate pair whole-genome sequencing to fine map rearrangement breakpoint junctions in both phenotypically normal and affected translocation carriers. In total, 46 junctions from 22 carriers of balanced translocations were characterized. Genes were disrupted in 48% of the breakpoints; recessive genes in four normal carriers and known dominant intellectual disability genes in three affected carriers. Finally, seven candidate disease genes were disrupted in five carriers with neurocognitive disabilities (SVOPL, SUSD1, TOX, NCALD, SLC4A10) and one XX-male carrier with Tourette syndrome (LYPD6, GPC5). Breakpoint junction analyses revealed microhomology and small templated insertions in a substantive fraction of the analyzed translocations (17.4%; n = 4); an observation that was substantiated by reanalysis of 37 previously published translocation junctions. Microhomology associated with templated insertions is a characteristic seen in the breakpoint junctions of rearrangements mediated by error-prone replication-based repair mechanisms. Our data implicate that a mechanism involving template switching might contribute to the formation of at least 15% of the interchromosomal translocation events.

  • 14. Richter, Karin
    et al.
    Wirta, Valtteri
    KTH, Skolan för bioteknologi (BIO).
    Dahl, Lina
    Bruce, Sara
    KTH, Skolan för bioteknologi (BIO).
    Lundeberg, Joakim
    KTH, Skolan för bioteknologi (BIO).
    Carlsson, Leif
    Williams, Cecilia
    KTH, Skolan för bioteknologi (BIO).
    Global gene expression analyses of hematopoietic stem cell-like cell lines with inducible Lhx2 expression2006Ingår i: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 7, s. 75-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Expression of the LIM-homeobox gene Lhx2 in murine hematopoietic cells allows for the generation of hematopoietic stem cell (HSC)-like cell lines. To address the molecular basis of Lhx2 function, we generated HSC-like cell lines where Lhx2 expression is regulated by a tet-on system and hence dependent on the presence of doxycyclin (dox). These cell lines efficiently down-regulate Lhx2 expression upon dox withdrawal leading to a rapid differentiation into various myeloid cell types.

    Results: Global gene expression of these cell lines cultured in dox was compared to different time points after dox withdrawal using microarray technology. We identified 267 differentially expressed genes. The majority of the genes overlapping with HSC-specific databases were those down-regulated after turning off Lhx2 expression and a majority of the genes overlapping with those defined as late progenitor-specific genes were the up-regulated genes, suggesting that these cell lines represent a relevant model system for normal HSCs also at the level of global gene expression. Moreover, in situ hybridisations of several genes down-regulated after dox withdrawal showed overlapping expression patterns with Lhx2 in various tissues during embryonic development.

    Conclusion: Global gene expression analysis of HSC-like cell lines with inducible Lhx2 expression has identified genes putatively linked to self-renewal / differentiation of HSCs, and function of Lhx2 in organ development and stem / progenitor cells of non-hematopoietic origin.

  • 15. Seidel, Sascha
    et al.
    Garvalov, Boyan K.
    Wirta, Valtteri
    KTH, Skolan för bioteknologi (BIO), Genteknologi.
    von Stechow, Louise
    Schaenzer, Anne
    Meletis, Konstantinos
    Wolter, Marietta
    Sommerlad, Daniel
    Henze, Anne-Theres
    Nister, Monica
    Reifenberger, Guido
    Lundeberg, Joakim
    KTH, Skolan för bioteknologi (BIO), Genteknologi.
    Frisen, Jonas
    Acker, Till
    A hypoxic niche regulates glioblastoma stem cells through hypoxia inducible factor 2 alpha2010Ingår i: Brain, ISSN 0006-8950, E-ISSN 1460-2156, Vol. 133, s. 983-995Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Glioma growth and progression depend on a specialized subpopulation of tumour cells, termed tumour stem cells. Thus, tumour stem cells represent a critical therapeutic target, but the molecular mechanisms that regulate them are poorly understood. Hypoxia plays a key role in tumour progression and in this study we provide evidence that the hypoxic tumour microenvironment also controls tumour stem cells. We define a detailed molecular signature of tumour stem cell genes, which are overexpressed by tumour cells in vascular and perinecrotic/hypoxic niches. Mechanistically, we show that hypoxia plays a key role in the regulation of the tumour stem cell phenotype through hypoxia-inducible factor 2 alpha and subsequent induction of specific tumour stem cell signature genes, including mastermind-like protein 3 (Notch pathway), nuclear factor of activated T cells 2 (calcineurin pathway) and aspartate beta-hydroxylase domain-containing protein 2. Notably, a number of these genes belong to pathways regulating the stem cell phenotype. Consistently, tumour stem cell signature genes are overexpressed in newly formed gliomas and are associated with worse clinical prognosis. We propose that tumour stem cells are maintained within a hypoxic niche, providing a functional link between the well-established role of hypoxia in stem cell and tumour biology. The identification of molecular regulators of tumour stem cells in the hypoxic niche points to specific signalling mechanisms that may be used to target the glioblastoma stem cell population.

  • 16.
    Sievertzon, Maria
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Wirta, Valtteri
    KTH, Skolan för bioteknologi (BIO).
    Mercer, Alex
    Frisén, Jonas
    Lundeberg, Joakim
    KTH, Skolan för bioteknologi (BIO), Genteknologi.
    Epidermal growth factor (EGF) withdrawal masks gene expression differences in the study of pituitary adenylate cyclase-activating polypeptide (PACAP) activation of primary neural stem cell proliferation2005Ingår i: BMC neuroscience (Online), ISSN 1471-2202, E-ISSN 1471-2202, Vol. 6, s. 55-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: The recently discovered adult neural stem cells, which maintain continuous generation of new neuronal and glial cells throughout adulthood, are a promising and expandable source of cells for use in cell replacement therapies within the central nervous system. These cells could either be induced to proliferate and differentiate endogenously, or expanded and differentiated in culture before being transplanted into the damaged site of the brain. In order to achieve these goals effective strategies to isolate, expand and differentiate neural stem cells into the desired specific phenotypes must be developed. However, little is known as yet about the factors and mechanisms influencing these processes. It has recently been reported that pituitary adenylate cyclase-activating polypeptide (PACAP) promotes neural stem cell proliferation both in vivo and in vitro.

    Results: We used cDNA microarrays with the aim of analysing the transcriptional changes underlying PACAP induced proliferation of neural stem cells. The primary neural stem/progenitor cells used were neurospheres, generated from the lateral ventricle wall of the adult mouse brain. The results were compared to both differentiation and proliferation controls, which revealed an unexpected and significant differential expression relating to withdrawal of epidermal growth factor (EGF) from the neurosphere growth medium. The effect of EGF removal was so pronounced that it masked the changes in gene expression patterns produced by the addition of PACAP.

    Conclusion: Experimental models aiming at transcriptional analysis of induced proliferation in primary neural stem cells need to take into consideration the significant effect on transcription caused by removal of EGF. Alternatively, EGF-free culture conditions need to be developed.

  • 17.
    Sievertzon, Maria
    et al.
    KTH, Skolan för bioteknologi (BIO), Centra, KTH Genome Center.
    Wirta, Valtteri
    KTH, Skolan för bioteknologi (BIO), Centra, KTH Genome Center.
    Mercer, Alex
    Meletis, Konstantinos
    Erlandsson, Rikard
    Wikström, Lilian
    Frisén, Jonas
    Lundeberg, Joakim
    KTH, Skolan för bioteknologi (BIO), Centra, KTH Genome Center.
    Transcriptome analysis in primary neural stem cells using a tag cDNA amplification method2005Ingår i: BMC neuroscience (Online), ISSN 1471-2202, E-ISSN 1471-2202, Vol. 6, nr 28, s. 13-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Neural stem cells ( NSCs) can be isolated from the adult mammalian brain and expanded in culture, in the form of cellular aggregates called neurospheres. Neurospheres provide an in vitro model for studying NSC behaviour and give information on the factors and mechanisms that govern their proliferation and differentiation. They are also a promising source for cell replacement therapies of the central nervous system. Neurospheres are complex structures consisting of several cell types of varying degrees of differentiation. One way of characterising neurospheres is to analyse their gene expression profiles. The value of such studies is however uncertain since they are heterogeneous structures and different populations of neurospheres may vary significantly in their gene expression.

    Results: To address this issue, we have used cDNA microarrays and a recently reported tag cDNA amplification method to analyse the gene expression profiles of neurospheres originating from separate isolations of the lateral ventricle wall of adult mice and passaged to varying degrees. Separate isolations as well as consecutive passages yield a high variability in gene expression while parallel cultures yield the lowest variability.

    Conclusions: We demonstrate a low technical amplification variability using the employed amplification strategy and conclude that neurospheres from the same isolation and passage are sufficiently similar to be used for comparative gene expression analysis.

  • 18. Stranneheim, Henrik
    et al.
    Engvall, Martin
    Naess, Karin
    Lesko, Nicole
    Larsson, Pontus
    Dahlberg, Mats
    Andeer, Robin
    Wredenberg, Anna
    Freyer, Chris
    Barbaro, Michela
    Bruhn, Helene
    Emahazion, Tesfail
    Magnusson, Måns
    Wibom, Rolf
    Zetterström, Rolf H.
    Wirta, Valtteri
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    von Döbeln, Ulrika
    Wedell, Anna
    Rapid pulsed whole genome sequencing for comprehensive acute diagnostics of inborn errors of metabolism2014Ingår i: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 15, s. 1090-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Massively parallel DNA sequencing (MPS) has the potential to revolutionize diagnostics, in particular for monogenic disorders. Inborn errors of metabolism (IEM) constitute a large group of monogenic disorders with highly variable clinical presentation, often with acute, nonspecific initial symptoms. In many cases irreversible damage can be reduced by initiation of specific treatment, provided that a correct molecular diagnosis can be rapidly obtained. MPS thus has the potential to significantly improve both diagnostics and outcome for affected patients in this highly specialized area of medicine. Results: We have developed a conceptually novel approach for acute MPS, by analysing pulsed whole genome sequence data in real time, using automated analysis combined with data reduction and parallelization. We applied this novel methodology to an in-house developed customized work flow enabling clinical-grade analysis of all IEM with a known genetic basis, represented by a database containing 474 disease genes which is continuously updated. As proof-of-concept, two patients were retrospectively analysed in whom diagnostics had previously been performed by conventional methods. The correct disease-causing mutations were identified and presented to the clinical team after 15 and 18 hours from start of sequencing, respectively. With this information available, correct treatment would have been possible significantly sooner, likely improving outcome. Conclusions: We have adapted MPS to fit into the dynamic, multidisciplinary work-flow of acute metabolic medicine. As the extent of irreversible damage in patients with IEM often correlates with timing and accuracy of management in early, critical disease stages, our novel methodology is predicted to improve patient outcome. All procedures have been designed such that they can be implemented in any technical setting and to any genetic disease area. The strategy conforms to international guidelines for clinical MPS, as only validated disease genes are investigated and as clinical specialists take responsibility for translation of results. As follow-up in patients without any known IEM, filters can be lifted and the full genome investigated, after genetic counselling and informed consent.

  • 19.
    Williams, Cecilia
    et al.
    KTH, Skolan för bioteknologi (BIO), Genteknologi.
    Wirta, Valtteri
    KTH, Skolan för bioteknologi (BIO), Genteknologi.
    Meletis, Konstantinos
    Wikström, Lilian
    Carlsson, Leif
    Frisén, Jonas
    Lundeberg, Joakim
    KTH, Skolan för bioteknologi (BIO), Genteknologi.
    Catalog of gene expression in adult neural stem cells and their in vivo microenvironment2006Ingår i: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 312, nr 10, s. 1798-1812Artikel i tidskrift (Refereegranskat)
    Abstract [en]

     Stem cells generally reside in a stem cell micro environment, where cues for self-renewal and differentiation are present. However, the genetic program underlying stem cell proliferation and multipotency is poorly understood. Transcriptome analysis of stem cells and their in vivo microenvironment is one way of uncovering the unique sternness properties and provides a framework for the elucidation of stem cell function. Here, we characterize the gene expression profile of the in vivo neural stem cell microenvironment in the lateral ventricle wall of adult mouse brain and of in vitro proliferating neural stem cells. We have also analyzed an Lhx2-expressing hematopoietic-stem-cell-like cell line in order to define the transcriptome of a well-characterized and pure cell population with stem cell characteristics. We report the generation, assembly and annotation of 50,792 high-quality 5'-end expressed sequence tag sequences. We further describe a shared expression of 1065 transcripts by all three stem cell libraries and a large overlap with previously published gene expression signatures for neural stem/progenitor cells and other multipotent stem cells. The sequences and cDNA clones obtained within this framework provide a comprehensive resource for the analysis of genes in adult stem cells that can accelerate future stem cell research.

  • 20.
    Wirta, Valtteri
    KTH, Skolan för bioteknologi (BIO).
    Mining the transcriptome - methods and applications2006Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Regulation of gene expression occupies a central role in the control of the flow of genetic information from genes to proteins. Regulatory events on multiple levels ensure that the majority of the genes are expressed under controlled circumstances to yield temporally controlled, cell and tissue-specific expression patterns. The combined set of expressed RNA transcripts constitutes the transcriptome of a cell, and can be analysed on a large-scale using both sequencing and microarray-based methods.

    The objective of this work has been to develop tools for analysis of the transcriptomes (methods), and to gain new insights into several aspects of the stem cell transcriptome (applications). During recent years expectations of stem cells as a resource for treatment of various disorders have emerged. The successful use of endogenously stimulated or ex vivo expanded stem cells in the clinic requires an understanding of mechanisms controlling their proliferation and self-renewal.

    This thesis describes the development of tools that facilitate analysis of minute amounts of stem cells, including RNA amplification methods and generation of a cDNA array enriched for genes expressed in neural stem cells. The results demonstrate that the proposed amplification method faithfully preserves the transcript expression pattern. An analysis of the feasibility of a neurosphere assay (in vitro model system for study of neural stem cells) clearly shows that the culturing induces changes that need to be taken into account in design of future comparative studies. An expressed sequence tag analysis of neural stem cells and their in vivo microenvironment is also presented, providing an unbiased large-scale screening of the neural stem cell transcriptome. In addition, molecular mechanisms underlying the control of stem cell self-renewal are investigated. One study identifies the proto-oncogene Trp53 (p53) as a negative regulator of neural stem cell self-renewal, while a second study identifies genes involved in the maintenance of the hematopoietic stem cell phenotype.

    To facilitate future analysis of neural stem cells, all microarray data generated is publicly available through the ArrayExpress microarray data repository, and the expressed sequence tag data is available through the GenBank.

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  • 21.
    Wirta, Valtteri
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Holmberg, Anders
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Lukacs, Morten
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Hilson, Pierre
    Uhlén, Mathias
    Bhalerao, Rishikesh
    Lundeberg, Joakim
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Assembly of a gene sequence tag microarray by reversible biotin-streptavidin capture for transcript analysis of Arabidopsis thaliana2005Ingår i: BMC Biotechnology, ISSN 1472-6750, E-ISSN 1472-6750, Vol. 5, s. 5-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Transcriptional profiling using microarrays has developed into a key molecular tool for the elucidation of gene function and gene regulation. Microarray platforms based on either oligonucleotides or purified amplification products have been utilised in parallel to produce large amounts of data. Irrespective of platform examined, the availability of genome sequence or a large number of representative expressed sequence tags ( ESTs) is, however, a pre-requisite for the design and selection of specific and high-quality microarray probes. This is of great importance for organisms, such as Arabidopsis thaliana, with a high number of duplicated genes, as cross-hybridisation signals between evolutionary related genes cannot be distinguished from true signals unless the probes are carefully designed to be specific.

    Results: We present an alternative solid-phase purification strategy suitable for efficient preparation of short, biotinylated and highly specific probes suitable for large-scale expression profiling. Twenty-one thousand Arabidopsis thaliana gene sequence tags were amplified and subsequently purified using the described technology. The use of the arrays is exemplified by analysis of gene expression changes caused by a four-hour indole-3-acetic ( auxin) treatment. A total of 270 genes were identified as differentially expressed ( 120 up-regulated and 150 down-regulated), including several previously known auxin-affected genes, but also several previously uncharacterised genes.

    Conclusions: The described solid-phase procedure can be used to prepare gene sequence tag microarrays based on short and specific amplified probes, facilitating the analysis of more than 21 000 Arabidopsis transcripts.

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