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  • 1.
    Badhai, Jitendra
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Fröjmark, Anne-Sophie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Razzaghian, Hamid Reza
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Davey, Edward
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Schuster, Jens
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Dahl, Niklas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Posttranscriptional down-regulation of small ribosomal subunit proteinscorrelates with reduction of 18S rRNA in RPS19 deficiency2009In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 583, no 12, p. 2049-2053Article in journal (Refereed)
    Abstract [en]

    Ribosomal protein S19 (RPS19) is mutated in patients with Diamond-Blackfan anemia (DBA). We hypothesized that decreased levels of RPS19 lead to a coordinated down-regulation of other ribosomal (r-)proteins at the subunit level. We show that small interfering RNA (siRNA) knock-down of RPS19 results in a relative decrease of small subunit (SSU) r-proteins (S20, S21 and S24) when compared to large subunit (LSU) r-proteins (L3, L9, L30 and L38). This correlates with a relative decrease in 18S rRNA with respect to 28S rRNA. The r-protein mRNA levels remain relatively unchanged indicating a post transcriptional regulation of r-proteins at the level of subunit formation.

  • 2. Barderi, P
    et al.
    Campetella, O
    Frasch, A C
    Santome, JA
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Pettersson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Cazzulo, JJ
    The NADP+ linked glutamate dehydrogenase from Trypanosoma cruzi: sequence, genomic organization and expression1998In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 330, no 2, p. 951-958Article in journal (Refereed)
    Abstract [en]

    NADP-linked glutamate dehydrogenase (NADP+-GluDH, EC 1.4.1.4) has been purified to homogeneity from epimastigotes of Trypanosoma cruzi by an improved procedure, and the amino acid sequences of 11 internal peptides obtained by digestion with trypsin, endopeptidase Lys-C, endopeptidase Arg-C or CNBr have been obtained. Using oligonucleotide primers synthesized according to the amino acid sequence of the N-terminus of the mature enzyme and to the nucleotide sequence of a clone corresponding to the C-terminus, obtained by immunological screening of an expression library, two complete open reading frames (TcGluDH1 and TcGluDH2) were isolated and sequenced. The sequences obtained are most similar to that of the NADP+-GluDH of Escherichia coli (70-72% identity), and less similar (50-56%) to those of lower eukaryotes. Using TcGluDH1 as a probe, evidence for the presence of several genes and developmental regulation of the expression of NADP+-GluDH in different parasite stages was obtained. TcGluDH1 encodes an enzymically active protein, since its expression in E. coli resulted in the production of a GluDH activity with kinetic parameters similar to those of the natural enzyme.

  • 3.
    Barrio, Alvaro Martinez
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Eriksson, Oskar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Badhai, Jitendra
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Fröjmark, Anne-Sophie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Bongcam-Rudloff, Erik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Dahl, Niklas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Schuster, Jens
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Targeted Resequencing and Analysis of the Diamond-Blackfan Anemia Disease Locus RPS192009In: PLoS ONE, ISSN 1932-6203, Vol. 4, no 7, p. e6172-Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: The Ribosomal protein S19 gene locus (RPS19) has been linked to two kinds of red cell aplasia, Diamond-Blackfan Anemia (DBA) and Transient Erythroblastopenia in Childhood (TEC). Mutations in RPS19 coding sequences have been found in 25% of DBA patients, but not in TEC patients. It has been suggested that non-coding RPS19 sequence variants contribute to the considerable clinical variability in red cell aplasia. We therefore aimed at identifying non-coding variations associated with DBA or TEC phenotypes. METHODOLOGY/PRINCIPAL FINDINGS: We targeted a region of 19'980 bp encompassing the RPS19 gene in a cohort of 89 DBA and TEC patients for resequencing. We provide here a catalog of the considerable, previously unrecognized degree of variation in this region. We identified 73 variations (65 SNPs, 8 indels) that all are located outside of the RPS19 open reading frame, and of which 67.1% are classified as novel. We hypothesize that specific alleles in non-coding regions of RPS19 could alter the binding of regulatory proteins or transcription factors. Therefore, we carried out an extensive analysis to identify transcription factor binding sites (TFBS). A series of putative interaction sites coincide with detected variants. Sixteen of the corresponding transcription factors are of particular interest, as they are housekeeping genes or show a direct link to hematopoiesis, tumorigenesis or leukemia (e.g. GATA-1/2, PU.1, MZF-1). CONCLUSIONS: Specific alleles at predicted TFBSs may alter the expression of RPS19, modify an important interaction between transcription factors with overlapping TFBS or remove an important stimulus for hematopoiesis. We suggest that the detected interactions are of importance for hematopoiesis and could provide new insights into individual response to treatment.

  • 4.
    Björck, Martin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Vascular Surgery.
    Pigg, Maritta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Kragsterman, Björn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Vascular Surgery.
    Bergqvist, David
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Vascular Surgery.
    Fatal bleeding following delivery: a manifestation of the vascular type of Ehlers-Danlos' syndrome2007In: Gynecologic and Obstetric Investigation, ISSN 0378-7346, E-ISSN 1423-002X, Vol. 63, no 3, p. 173-175Article in journal (Refereed)
    Abstract [en]

    Introduction: The vascular form of Ehlers-Danlos' syndrome (type IV) is a potentially lethal genetic condition because of rupture of major arteries, often in the peri-partum period. Case Report: We report a 31-year-old primipara who died from a rupture of the right subclavian artery. The patient had several symptoms and signs typical of the disease. The rupture occurred during the expulsion-phase of delivery but was recognized only on day 9. Conclusion: Early recognition is crucial to avoid maternal mortality due to this genetic disorder. Once the condition is suspected, the clinical diagnosis is straightforward.

  • 5. Bontempi, EJ
    et al.
    Garcia, GA
    Buschiazzo, A
    Henriksson, Jan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Pravia, CA
    Ruiz, AM
    Pettersson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Pszenny, V
    The tyrosine aminotransferase from Trypanosoma rangeli: sequence andgenomic characterization2000In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 189, no 2, p. 253-257Article in journal (Refereed)
    Abstract [en]

    The complete sequence and genomic characterization of the tyrosine aminotransferase (TAT) gene from Trypanosoma rangeli is reported. The gene was found to be organized in a tandem multicopy gene array. A homologous mRNA species (2.5 kb) was identified in the epimastigote form of the parasite. From the deduced amino acid sequence, the gene encodes a protein of 420 amino acids with a predicted molecular mass of 46.4 kDa and a theoretical pI of 6.23. A high sequence identity was found with the Trypanosoma cruzi, human and rat enzymes. All the essential residues for TAT enzymatic activity are conserved, as well as a pyridoxal-phosphate attachment site typical of class-I aminotransferases. The recombinant enzyme was recognized by a monoclonal antibody against the T. cruzi enzyme. Additionally, the recombinant protein showed enzymatic activity when incubated with L-tyrosine and 2-oxoglutaric acid as substrates.

  • 6.
    Bridge, Eileen
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Riedel, Kai-Uwe
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Johansson, Britt-Marie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Pettersson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Spliced exons of adenovirus late RNAs colocalize with snRNP in a specific nuclear domain1996In: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 135, no 2, p. 303-314Article in journal (Refereed)
    Abstract [en]

    Posttranscriptional steps in the production of mRNA include well characterized polyadenylation and splicing reactions, but it is also necessary to understand how RNA is transported within the nucleus from the site of its transcription to the nuclear pore, where it is translocated to the cytoplasmic compartment. Determining the localization of RNA within the nucleus is an important aspect of understanding RNA production and may provide clues for investigating the trafficking of RNA within the nucleus and the mechanism for its export to the cytoplasm. We have previously shown that late phase adenovirus-infected cells contain large clusters of snRNP and non-snRNP splicing factors; the presence of these structures is correlated with high levels of viral late gene transcription. The snRNP clusters correspond to enlarged interchromatin granules present in late phase infected cells. Here we show that polyadenylated RNA and spliced tripartite leader exons from the viral major late transcription unit are present in these same late phase snRNP-containing structures. We find that the majority of the steady state viral RNA present in the nucleus is spliced at the tripartite leader exons. Tripartite leader exons are efficiently exported from the nucleus at a time when we detect their accumulation in interchromatin granule clusters. Since the enlarged interchromatin granules contain spliced and polyadenylated RNA, we suggest that viral RNA may accumulate in this late phase structure during an intranuclear step in RNA transport.

  • 7.
    Bridge, Eileen
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Xia, Dong-Xiang
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Carmo-Fonseca, M
    Cardinali, B
    Lamond, A I
    Pettersson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Dynamic organization of splicing factors in adenovirus-infected cells1995In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 69, no 1, p. 281-290Article in journal (Refereed)
    Abstract [en]

    Adenovirus infection affects the nuclear distribution of host splicing factors. Late phase-infected cells contain discrete clusters of small nuclear ribonucleoproteins (snRNPs) that are separate from centers containing the viral 72-kilodalton DNA-binding protein (72K protein). In the present study, we demonstrate that these snRNP clusters also contain splicing factors from the SR protein family. We show that a previously described monoclonal antibody, 3C5, detects SR proteins. Furthermore, we demonstrate that late region 3 transcription occurs at a maximal rate in infected cultures in which greater than 90% of the cells contain the snRNP clusters, indicating that such cells are actively transcribing their late genes. During the onset of the late phase, the intranuclear distribution of splicing factors is very different from that seen after the late phase is established. When late viral transcription commences, cells with snRNP clusters are less prevalent than in cultures that are maintaining maximum levels of late transcription. Instead, a cell type which shows snRNPs, concentrated in foci that also contain the viral 72K DNA-binding protein is detected. This cell type disappears from cultures by 18 to 20 h after a high-multiplicity infection. These results suggest a dynamic organization of splicing factors in infected cells that can be correlated to the status of viral gene expression. Our work also provides an explanation for the differing results that have been published concerning the organization of splicing factors in the adenovirus-infected cell nucleus (L. F. Jiménez-García and D. L. Spector, Cell 73:47-59, 1993). During the present study we observed that a monoclonal antibody against the SC-35 protein, which was used by Jiménez-García and Spector to study the localization of the SC-35 splicing factor in adenovirus-infected cells, cross-reacts with the adenovirus 72K DNA-binding protein and is thus unsuitable for this type of study.

  • 8.
    Buckley, Patrick G
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Mantripragada, Kiran K
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Benetkiewicz, Magdalena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Tapia-Paez, Isabel
    Diaz de Ståhl, Teresita
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Rosenquist, Magnus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Ali, Haider
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Jarbo, Caroline
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    de Bustos, Cecilía
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Hirvela, Carina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Sinder Wilén, Birgitta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Fransson, Ingegerd
    Thyr, Charlotte
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Johnsson, Britt-Inger
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Bruder, Carl E G
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Menzel, Uwe
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Hergersberg, Martin
    Mandahl, Nils
    Blennow, Elisabeth
    Wedell, Anna
    Beare, David M
    Collins, John E
    Dunham, Ian
    Albertson, Donna
    Pinkel, Daniel
    Bastian, Boris C
    Faruqi, A Fawad
    Lasken, Roger S
    Ichimura, Koichi
    Collins, V Peter
    Dumanski, Jan P
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    A full-coverage, high-resolution human chromosome 22 genomic microarrayfor clinical and research applications2002In: Human Molecular Genetics, ISSN 0964-6906, E-ISSN 1460-2083, Vol. 11, no 25, p. 3221-3229Article in journal (Refereed)
    Abstract [en]

    We have constructed the first comprehensive microarray representing a human chromosome for analysis of DNA copy number variation. This chromosome 22 array covers 34.7 Mb, representing 1.1% of the genome, with an average resolution of 75 kb. To demonstrate the utility of the array, we have applied it to profile acral melanoma, dermatofibrosarcoma, DiGeorge syndrome and neurofibromatosis 2. We accurately diagnosed homozygous/heterozygous deletions, amplifications/gains, IGLV/IGLC locus instability, and breakpoints of an imbalanced translocation. We further identified the 14-3-3 eta isoform as a candidate tumor suppressor in glioblastoma. Two significant methodological advances in array construction were also developed and validated. These include a strictly sequence defined, repeat-free, and non-redundant strategy for array preparation. This approach allows an increase in array resolution and analysis of any locus; disregarding common repeats, genomic clone availability and sequence redundancy. In addition, we report that the application of phi29 DNA polymerase is advantageous in microarray preparation. A broad spectrum of issues in medical research and diagnostics can be approached using the array. This well annotated and gene-rich autosome contains numerous uncharacterized disease genes. It is therefore crucial to associate these genes to specific 22q-related conditions and this array will be instrumental towards this goal. Furthermore, comprehensive epigenetic profiling of 22q-located genes and high-resolution analysis of replication timing across the entire chromosome can be studied using our array.

  • 9. Carvalho, T
    et al.
    Seeler, J S
    Ohman, K
    Jordan, P
    Pettersson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Akusjärvi, Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Carmo-Fonseca, M
    Dejean, A
    Targeting of adenovirus E1A and E4-ORF3 proteins to nuclear matrix-associated PML bodies1995In: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 131, no 1, p. 45-56Article in journal (Refereed)
    Abstract [en]

    The PML protein was first identified as part of a fusion product with the retinoic acid receptor alpha (RAR alpha), resulting from the t(15;17) chromosomal translocation associated with acute promyelocytic leukemia (APL). It has been previously demonstrated that PML, which is tightly bound to the nuclear matrix, concentrates in discrete subnuclear compartments that are disorganized in APL cells due to the expression of the PML-RAR alpha hybrid. Here we report that adenovirus infection causes a drastic redistribution of PML from spherical nuclear bodies into fibrous structures. The product encoded by adenovirus E4-ORF3 is shown to be responsible for this reorganization and to colocalize with PML into these fibers. In addition, we demonstrate that E1A oncoproteins concentrate in the PML domains, both in infected and transiently transfected cells, and that this association requires the conserved amino acid motif (D)LXCXE, common to all viral oncoproteins that bind pRB or the related p107 and p130 proteins. The SV-40 large T antigen, another member of this oncoprotein family is also found in close association with the PML nuclear bodies. Taken together, the present data indicate that the subnuclear domains containing PML represent a preferential target for DNA tumor viruses, and therefore suggest a more general involvement of the PML nuclear bodies in oncogenic processes.

  • 10. Chen, Richard Z
    et al.
    Pettersson, Ulf
    Whitehead Institute for Biomedical Research, Department of Biology, Cambridge, USA.
    Beard, Caroline
    Jackson-Grusby, Laurie
    Jaenisch, Rudolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    DNA hypomethylation leads to elevated mutation rates1998In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 395, no 6697, p. 89-93Article in journal (Refereed)
    Abstract [en]

    Genome-wide demethylation has been suggested to be a step in carcinogenesis. Evidence for this notion comes from the frequently observed global DNA hypomethylation in tumour cells, and from a recent study suggesting that defects in DNA methylation might contribute to the genomic instability of some colorectal tumour cell lines. DNA hypomethylation has also been associated with abnormal chromosomal structures, as observed in cells from patients with ICF (Immunodeficiency, Centromeric instability and Facial abnormalities) syndrome and in cells treated with the demethylating agent 5-azadeoxycytidine. Here we report that murine embryonic stem cells nullizygous for the major DNA methyltransferase (Dnmt1) gene exhibited significantly elevated mutation rates at both the endogenous hypoxanthine phosphoribosyltransferase (Hprt) gene and an integrated viral thymidine kinase (tk) transgene. Gene deletions were the predominant mutations at both loci. The major cause of the observed tk deletions was either mitotic recombination or chromosomal loss accompanied by duplication of the remaining chromosome. Our results imply an important role for mammalian DNA methylation in maintaining genome stability.

  • 11.
    Dahl, Niklas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Segmentella DNA-variationer driver genomets evolution. Ger nya infallsvinklar till förståelse av sjukdom och hälsa: [Segmental DNA variations impel the genome evolution. New approaches to the understanding of disease and health]2010In: Läkartidningen, ISSN 0023-7205, E-ISSN 1652-7518, Vol. 107, no 17, p. 1138-1139Article in journal (Other academic)
  • 12.
    Edman Ahlbom, Bodil
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Goetz, P
    Korenberg, JR
    Pettersson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Seemenova, E
    Wadelius, Claes
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Zech, Lore
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Annerén, Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Molecular analysis of chromosome 21 in a patient with a phenotype of down syndrome and apparently normal karyotype1996In: American Journal of Medical Genetics. Part A, ISSN 1552-4825, E-ISSN 1552-4833, Vol. 63, no 4, p. 566-572Article in journal (Refereed)
    Abstract [en]

    Down syndrome (DS) is caused in most cases by the presence of an extra chromosome 21. It has been shown that the DS phenotype is produced by duplication of only a small part of the long arm of chromosome 21, the 21q22 region, including and distal to locus D21S55. We present molecular investigations on a woman with clinically typical DS but apparently normal chromosomes. Her parents were consanguineous and she had a sister with a DS phenotype, who died at the age of 15 days. Repeated cytogenetic investigations (G-banding and high resolution banding) on the patient and her parents showed apparently normal chromosomes. Autoradiographs of quantitative Southern blots of DNAs from the patient, her parents, trisomy 21 patients, and normal controls were analyzed after hybridization with unique DNA sequences regionally mapped on chromosome 21. Sequences D21S59, D21S1, D21S11, D21S8, D21S17, D21S55, ERG, D21S15, D21S112, and COL6A1 were all found in two copies. Fluorescent in situ hybridization with a chromosome 21-specific genomic library showed no abnormalities and only two copies of chromosome 21 were detected. Nineteen markers from the critical region studied with polymerase chain reaction amplification of di- and tetranucleotide repeats did not indicate any partial trisomy 21. From this study we conclude that the patient does not have any partial submicroscopic trisomy for any segment of chromosome 21. It seems reasonable to assume that she suffers from an autosomal recessive disorder which is phenotypically indistinguishable from DS.

  • 13.
    Fredriksson, Mona
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Barbany, Gisela
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Liljedahl, Ulrika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Molecular Medicine.
    Hermanson, Monica
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Kataja, Matti
    Syvänen, Ann-Christine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Molecular Medicine.
    Assessing hematopoietic chimerism after stem cell transplantation by multiplexed SNP genotyping using microarrays and quantitative analysis of SNP alleles2004In: Leukemia, ISSN 0887-6924, E-ISSN 1476-5551, Vol. 18, no 2, p. 255-266Article in journal (Refereed)
    Abstract [en]

    Single-nucleotide polymorphisms (SNPs) have the potential to be particularly useful as markers for monitoring of chimerism after stem cell transplantation (SCT) because they can be analyzed by accurate and robust methods. We used a two-phased minisequencing strategy for monitoring chimerism after SCT. First, informative SNPs with alleles differing between donor and recipient were identified using a multiplex microarray-based minisequencing system screening 51 SNPs to ensure that multiple informative SNPs were detected in each donor-recipient pair. Secondly, the development of chimerism was followed up after SCT by sensitive, quantitative analysis of individual informative SNPs by applying the minisequencing method in a microtiter plate format. Using this panel of SNPs, we identified multiple informative SNPs in nine unrelated and in 16 related donor-recipient pairs. Samples from nine of the donor-recipient pairs taken at time points ranging from 1 month to 8 years after transplantation were available for analysis. In these samples, we monitored the allelic ratios of two or three informative SNPs in individual minisequencing reactions. The results agreed well with the data obtained by microsatellite analysis. Thus, we conclude that the two-phased minisequencing strategy is a useful approach in the following up of patients after SCT.

  • 14.
    Fröjmark, Anne-Sophie
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Badhai, Jitendra
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Klar, Joakim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Thuveson, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Schuster, Jens
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Dahl, Niklas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Cooperative effect of ribosomal protein s19 and Pim-1 kinase on murine c-Myc expression and myeloid/erythroid cellularity2010In: Journal of Molecular Medicine, ISSN 0946-2716, E-ISSN 1432-1440, Vol. 88, no 1, p. 39-46Article in journal (Refereed)
    Abstract [en]

    Diamond Blackfan anemia (DBA) is a bone marrow failure syndrome associated with heterozygous mutations in the ribosomal protein S19 (RPS19) gene in a subgroup of patients. One of the interacting partners with RPS19 is the oncoprotein PIM-1 kinase. We intercrossed Rps19+/- and Pim-1-/- mice strains to study the effect from the disruption of both genes. The double mutant (Rps19+/-Pim-1-/-) mice display normal growth with increased peripheral white- and red blood cell counts when compared to the w.t. mice (Rps19+/+Pim-1+/+). Molecular analysis of bone marrow cells in Rps19+/-Pim-1-/- mice revealed up-regulated levels of c-Myc and the anti-apoptotic factors Bcl2, BclXL and Mcl-1. This is associated with a reduction of the apoptotic factors Bak and Caspase 3 as well as the cell cycle regulator p21. Our findings suggest that combined Rps19 insufficiency and Pim-1 deficiency promote murine myeloid cell growth through a deregulation of c-Myc and a simultaneous up-regulation of anti-apoptotic Bcl proteins.

  • 15.
    Hallböök, Finn
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Ebendal, Ted
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Zoology.
    Persson, Håkan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Production and characterization of biologically active recombinant beta nerve growth factor1988In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 8, no 1, p. 452-456Article in journal (Refereed)
    Abstract [en]

    DNA fragments encoding either rat or chicken beta nerve growth factor (NGF) were inserted in the expression vector p91023(B) for transient expression in COS cells. The two NGF constructs produced RNA transcripts and proteins of the predicted sizes. Conditioned media from the transfected cells stimulated neurite outgrowth from cultured chicken embryo sympathetic ganglia. The results show that the rat or chicken NGF gene can direct the synthesis of a biologically active NGF protein after transfection of COS cells.

  • 16.
    Henriksson, J
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Dujardin, JC
    Barnabe, C
    Brisse, S
    Timperman, G
    Venegas, J
    Pettersson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Tibayrenc, M
    Solari, A
    Chromosomal size variation in Trypanosoma cruzi is mainly progressive andis evolutionarily informative2002In: Parasitology, ISSN 0031-1820, E-ISSN 1469-8161, Vol. 124, no Pt3, p. 277-286Article in journal (Refereed)
    Abstract [en]

    The evolutionary significance of chromosome size polymorphism was explored in a representative panel of 26 Trypanosoma cruzi stocks. We tested a progressive model (aCSDI) assuming that the larger the size difference between homologous chromosomes, the more divergent the parasites are. This was contrasted with a non-progressive model (Jaccard's distance), in which any chromosome size difference has the same weight. ACSDI-based dendrograms were very similar to those built-up from multilocus enzyme electrophoresis (MLEE) and random amplified polymorphic DNA (RAPD) data: structuring in 2 major lineages (T. cruzi I and T. cruz II) and 5 small subdivisions within T. cruzi II was identical, and branching was very similar. Furthermore, a significant correlation (P < 0.001) was observed between aCSDI and phenetic distances calculated from MLEE and RAPD data. In contrast, analysis of chromosome size polymorphism with Jaccard's distance generated dendrograms with relatively long branches, causing most branching points to cluster close together, which generates statistically uncertain branching points. Our results thus support a model of progressive chromosome size-variation and show that despite an extensive polymorphism, chromosomal sizes constitute valuable characters for evolutionary analyses. Furthermore, our data are consistent with the clonal evolution model previously proposed for T. cruzi.

  • 17.
    Henriksson, Jan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Porcel, Betina
    Rydåker, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Ruiz, A
    Sabaj, V
    Galanti, Norbel
    Cazzulo, J J
    Frasch, A C
    Pettersson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Chromosome specific markers reveal conserved linkage groups in spite of extensive chromosomal size variation in Trypanosoma cruzi1995In: Molecular and biochemical parasitology (Print), ISSN 0166-6851, E-ISSN 1872-9428, Vol. 73, no 1-2, p. 63-74Article in journal (Refereed)
    Abstract [en]

    The karyotypes of three cloned stocks, CL Brener (CL), CA I/72 (CA) and Sylvio X10/7 (X10), of Trypanosoma cruzi were studied by pulsed-field gel electrophoresis followed by ethidium bromide staining and hybridization with 35 different probes, 30 of which identified single chromosomes. The chromosome-specific probes identified between 26 and 31 chromosomal bands in the three cloned stocks, corresponding to 20 unique chromosomes in CL and 19 in CA and X10. Considering the DNA content of the parasite, it was predicted that the markers recognise at least half of all T. cruzi chromosomes. A majority of identified chromosomes showed large differences in size among different strains, in some cases by up to 50%. Interestingly, CL had in general larger chromosomes than the two other studied cloned stocks. Several of the markers showed linkage and nine different linkage groups were identified, each comprising 2-4 markers. The linkage between the markers was maintained in 8 of the 9 linkage groups when a panel comprising 26 different T. cruzi strains representing major T. cruzi populations was tested. One linkage group was found to be maintained in some strains but not in others. This result shows that chromosomal rearrangements occur in the T. cruzi genome, albeit with a low frequency. Repetitive DNA, both non-coding and in one case coding, was more abundant in the cloned stock CL Brener than in CA and X10. The information presented will make it possible to select chromosomes for the construction of physical chromosomal maps required for the T. cruzi genome project.

  • 18.
    Henriksson, Jan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Åslund, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Pettersson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Karyotype variability in Trypanosoma cruzi1996In: Parasitology Today, ISSN 0169-4758, E-ISSN 1873-1473, Vol. 12, no 3, p. 108-114Article in journal (Refereed)
    Abstract [en]

    Like many other protozoam parasites, Trypanosoma cruzi (the causative agent of Chagas disease) has a plastic genome. Chromosome size polymorphisms occur in different strains of T. cruzi as well as among clones originating from the same strain, Despite this polymorphism, major interchromosomal rearrangements appear to be rare since several linkage groups of chromosomal markers are well conserved among different T. cruzi strains. In addition, some correlation has been found between karyotype variability and classification by multilocus enzyme electrophoresis. In this review, Jan Henriksson, Lena Åslund and Ulf Petterson discuss the genomic variability and suggest that amplication of repetitive sequences or members of gene families make a major contribution to the chromosomal size variation

  • 19.
    Jazin, Elena
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Bontempi, Esteban
    INDIECH, Av. Paseo Colón 568, (1063), Buenos Aires, Argentina.
    Sanchez, Daniel
    Åslund, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Henriksson, Jan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Frasch, Alberto
    Pettersson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Trypanosoma cruzi exoantigen is a member of a 160 kDa gene family1995In: Parasitology, ISSN 0031-1820, E-ISSN 1469-8161, Vol. 110, no Pt1, p. 61-69Article in journal (Refereed)
    Abstract [en]

    During the chronic stage of Chagas disease a 160 kDa antigen appears in the blood of patients and remains detectable many years after the onset of the disease. This antigen is secreted by the trypomastigote form of the parasite while it is undetectable in the epimastigote form. We report here that the chronic 160 kDa exoantigen is encoded by a gene family (CEA 160 family). We describe the cloning and partial nucleotide sequence of a gene (CEA 160-1) belonging to the CEA160 family. Comparison of the gene sequence with other sequences present in the databases revealed homologies with several Trypanosoma cruzi surface antigens. Highest amino acid identity (59%) was with members of a family containing epitopes that mimic nervous tissues (Van Voorhis et al. 1993). Another related group (18-22% amino acid identity) comprises proteins of 85 or 160 kDa sharing an amino acid motif that is conserved among bacterial neuraminidases (Fouts et al. 1991; Pollevick et al. 1991; Kahn et al. 1991; Takle & Cross, 1991; Franco et al. 1993). The amino acid identities with the different antigens were not homogeneously distributed. Regions of higher identity (40-60%) were grouped in the central region of each protein.

  • 20.
    Johansson, Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Microfluidic and Molecular Tools for Genetic Analyses2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Methods that enable interrogation of multiple genomic regions in parallel are very useful for efficient detection of genetic variation. Two different types of probes are described in this thesis that can be used for direct analysis or for sample preparation upstream of Next Generation Sequencing.  In addition to the development of molecular probing systems it also reports on the progress of two assay formats for biological experiments.

    The Selector probe enrich for genomic regions of interest by probe mediated specific circularization of target fragments. Amplification based enrichment of circles can be carried out using polymerase chain reaction, rolling-circle amplification or multiple displacement amplification. Enrichment of all exons in 28 genes known to be mutated in lung and/or colon cancer is demonstrated.  Selection and analysis by SOLiD Sequencing was performed on fresh frozen and formalin fixed paraffin embedded (FFPE) samples, and mutations previously detected by Sanger sequencing were detected.  The extractor probe is another probe variant that can be used for multiplex enrichment of DNA. It targets genomic fragments by using both ligation and sequence specific elongation for discrimination between on and off target sequences.

    A microfluidic platform fabricated by compact disc injection molding that can be used for biological assays is described.  Microchannel structures in thermoplastic material are coated with silicon dioxide by electron beam evaporation which facilitates closing of the structures by PDMS- glass bonding by ozone plasma. The platform’s utility for biological experiments is demonstrated by for detection of amplified single molecules (ASM), cell culturing and on-chip peristaltic pumping.

    The thesis also includes an exploratory study for the purpose of using a non-optical system for detection of ASM’s.  Optimizations were performed of the conditions needed in order to detect an increase in hydrodynamic size of magnetic particles, using a superconducting quantum interference device (SQUID), as they form complex with ASM’s.

     

    List of papers
    1. Targeted resequencing of candidate genes using Selector Probes
    Open this publication in new window or tab >>Targeted resequencing of candidate genes using Selector Probes
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    2011 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, no 2, p. e8-Article in journal (Refereed) Published
    Abstract [en]

    Targeted genome enrichment is a powerful tool for making use of the massive throughput of novel DNA-sequencing instruments. We herein present a simple and scalable protocol for multiplex amplification of target regions based on the Selector technique. The updated version exhibits improved coverage and compatibility with next-generation-sequencing (NGS) library-construction procedures for shotgun sequencing with NGS platforms. To demonstrate the performance of the technique, all 501 exons from 28 genes frequently involved in cancer were enriched for and sequenced in specimens derived from cell lines and tumor biopsies. DNA from both fresh frozen and formalin-fixed paraffin-embedded biopsies were analyzed and 94 specificity and 98 coverage of the targeted region was achieved. Reproducibility between replicates was high (R 2=0, 98) and readily enabled detection of copy-number variations. The procedure can be carried out in <24 h and does not require any dedicated instrumentation.

    Keywords
    Resequencing, Targeted, Next generation sequencing, Selectors, Cancer, Tumor, FFPE
    National Category
    Medical and Health Sciences
    Research subject
    Medical Genetics
    Identifiers
    urn:nbn:se:uu:diva-121425 (URN)10.1093/nar/gkq1005 (DOI)000286675300003 ()21059679 (PubMedID)
    Available from: 2010-03-23 Created: 2010-03-23 Last updated: 2017-12-12Bibliographically approved
    2. Multiplex PCR amplification using Extractor probes
    Open this publication in new window or tab >>Multiplex PCR amplification using Extractor probes
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    Manipulation of a plurality of DNA molecules in parallel offers several advantages over simplex reactions. This paper reports on a new version of Selector probes with easier design procedure and the possibility to achieve higher ROI content within the targeted fragments. To demonstrate its utility we applied it for copy number variance detection and simultaneous amplification of 64 genomic fragments.

    Keywords
    PCR, sequencing, genetics, CNV
    National Category
    Medical Genetics
    Research subject
    Genetics
    Identifiers
    urn:nbn:se:uu:diva-121532 (URN)
    Available from: 2010-03-24 Created: 2010-03-24 Last updated: 2018-01-12
    3. Interbead interactions within oligonucleotide functionalized ferrofluids suitable for magnetic biosensor applications
    Open this publication in new window or tab >>Interbead interactions within oligonucleotide functionalized ferrofluids suitable for magnetic biosensor applications
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    2007 (English)In: Journal of Physics D: Applied Physics, ISSN 0022-3727, E-ISSN 1361-6463, Vol. 40, no 5, p. 1320-1330Article in journal (Refereed) Published
    Abstract [en]

    Interactions in oligonucleotide functionalized ferrofluids were investigated by measurements of frequency dependent complex magnetization in a superconducting quantum interference device. The ferrofluids consisted of aqueous suspensions of single-stranded oligonucleotide functionalized 130 nm sized magnetic beads, suitable for further use in magnetic biosensor applications based on changes in the Brownian relaxation behaviour of oligonucleotide-coated beads. The interbead interactions were found to depend strongly on the surface coverage by oligonucleotide molecules. At low surface coverage, aggregates of beads within the ferrofluid were formed, most likely due to interbead SS-crosslinking reactions. When the surface coverage increased, the entropic interbead repulsion arising from the thermal motion of oligonucleotide molecules began to stabilize the ferrofluid by preventing aggregation, and the crosslinking probability decreased. At a surface coverage, of ~40 oligonucleotides per bead, determined by a radioactive labelling analysis, an optimal configuration was obtained for which the ferrofluid behaved as consisting of almost non-interacting beads. At higher oligonucleotide functionalization degrees, the high coverage induced less thermal motion flexibility of the oligonucleotide chains which decreased the entropic repulsion effect. This in turn led to a higher crosslinking probability, as well as an increase in the hydrodynamic size of the beads. Thus, the ferrofluid sample of nearly non-interacting nature, in which an optimal degree of stabilization and the highest Brownian relaxation frequency are achieved in the low surface coverage region, may be considered as the most appropriate one for further use in magnetic biosensor applications, from both a functional and an economic point of view.

    National Category
    Engineering and Technology
    Research subject
    Engineering Science with specialization in Nanotechnology and Functional Materials
    Identifiers
    urn:nbn:se:uu:diva-98019 (URN)10.1088/0022-3727/40/5/002 (DOI)000245283100022 ()
    Available from: 2009-02-13 Created: 2009-02-13 Last updated: 2017-12-14Bibliographically approved
    4. Thermoplastic Microfluidic Platform for Single-Molecule Detection, Cell Culture and Actuation
    Open this publication in new window or tab >>Thermoplastic Microfluidic Platform for Single-Molecule Detection, Cell Culture and Actuation
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    2005 In: Analytical Chemistry, ISSN 0003-2700, Vol. 77, no 22, p. 7122-7130Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-95117 (URN)
    Available from: 2006-11-22 Created: 2006-11-22 Last updated: 2010-03-25Bibliographically approved
  • 21.
    Karsten, Stanislav L.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Lagerstedt, Kristina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Carlberg, Britt-Marie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Kleijer, Wim J
    Zaremba, Jacek
    van Diggelen, Otto
    Czartoryska, Barbara
    Pettersson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Bondeson, Marie-Louise
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Two distinct deletions in the IDS gene and the gene W: a novel type of mutation associated with the Hunter syndrome1997In: Genomics, ISSN 0888-7543, E-ISSN 1089-8646, Vol. 43, no 2, p. 123-129Article in journal (Refereed)
    Abstract [en]

    A novel mutation has been identified in a patient with the Hunter syndrome (mucopolysaccharidosis type II), in whom the disorder is associated with two distinct deletions separated by 30 kb. The deletions were characterized by Southern blot and PCR analyses, and the nucleotide sequences at both junctions were determined. The first deletion, corresponding to a loss of 3152 bp of DNA, included exons 5 and 6 of the iduronate-2-sulfatase (IDS) gene. The second deletion was 3603 bp long and included exons 3 and 4 of geneW, which is located in the DXS466 locus telomeric of theIDSgene. Both deletions are the result of nonhomologous (illegitimate) recombination events between short direct repeats at the deletion breakpoints. An interesting finding was the presence of the heptamer sequence 5′-TACTCTA-3′ present at both deletion junctions, suggesting that this motif might be a hot spot for recombination. We propose that the double deletion is the result of homology-associated nonhomologous recombinations caused by the presence of large duplicated regions in Xq27.3–q28.

  • 22.
    Kirsebom, Leif A
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Virtanen, Anders
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    At the interface of RNA and protein: RNA-Protein Interactions (a book review)1995In: Trends in Cell Biolology, ISSN 0962-8924, E-ISSN 1879-3088, Vol. 5, no 12, p. 476-476Article, book review (Other academic)
  • 23.
    Kristjánsdóttir, Helga
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    The PD-1 pathway and the complement system in systemic lupus erythematosus2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Autoimmune diseases occur in up to 3-5% of the general population and represent a diverse collection of diseases with regards to clinical manifestations. The unifying factor of autoimmune diseases is tissue and organ damage as a result of an immune response mounted against self-antigens.

    Systemic lupus erythematosus (SLE) is considered a prototype of human systemic autoimmune diseases. The etiology of SLE is as yet largely unknown, but both epidemiological and genetic data suggest an interplay between numerous and varying genetic and environmental factors.

    There is compelling evidence for a strong genetic component in SLE. The disease has a high λsibs value and familial clustering is apparent. Multiple susceptibility loci have been identified, some of which are syntenic between humans and mice and some of which overlap with other autoimmune diseases.

     

    This thesis is based on analysis of Icelandic multicase SLE families and Swedish SLE patients.

    Paper I is a study of the association of C4A protein deficiency (C4AQ0) with SLE in the multicase families and shows a significantly increased frequency of C4AQ0 in the families. The genetic basis for C4AQ0 varies and C4AQ0 is found on different MHC haplotypes, pointing to C4AQ0 as an independent risk factor for SLE.

    Paper II describes the association of low MBL serum levels with SLE in the families and identifies low MBL as risk factor for SLE in families that carry the defect. Low MBL was furthermore found to mediate an additive risk when found in combination with C4AQ0.

    In paper III cellular expression the PD-1 co-inhibitory receptor on T cells was studied. A polymorphism in the PDCD1 gene, PD-1.3A was previously associated with SLE in the multicase families. The polymorphism is thought to disrupt expression of the gene and may lead to decreased expression of the PD-1 receptor. The study demonstrates lower PD-1 expression in SLE patients and relatives in correlation to the PD-1.3A genotype.

    Paper IV is a compiled analysis of the SLE families, including PD-1.3A, C4AQ0, low MBL, autoimmune diseases and autoantibody profiles. The study demonstrates clustering of different autoimmune diseases and autoantibodies in families that are heterogenic with regards to the genetic susceptibility factors, PD-1.3A, C4AQ0 and low MBL.

    List of papers
    1. A study of the genetic basis of C4A protein deficiency. Detection of C4A gene deletion by long-range PCR and its associated haplotypes.
    Open this publication in new window or tab >>A study of the genetic basis of C4A protein deficiency. Detection of C4A gene deletion by long-range PCR and its associated haplotypes.
    2004 (English)In: Scandinavian Journal of Rheumatology, ISSN 0300-9742, E-ISSN 1502-7732, Vol. 33, no 6, p. 417-22Article in journal (Refereed) Published
    Abstract [en]

    Objective: Study the frequency of C4A gene deletions as the genetic basis of C4A protein deficiency (C4AQ0) and its associated haplotypes in Icelandic SLE families.

    Materials and methods: Nine multiplex SLE families were genotyped for C4A gene deletions using LR-PCR and MHC haplotypes were defined.

    Results: Of SLE patients, first- and second-degree relatives, 53,8%, 47,9% and 28,6% had C4AQ0, respectively. A C4A gene deletion is the genetic basis for C4AQ0 in 64,3% of SLE patients, 60,0% of first-degree and 50,0% of second-degree relatives.

    All individuals carrying haplotype B8-C4AQ0-C4B1-DR3 had a deletion and the deletion was also found on haplotypes B8-C4AQ0-C4B1-DR7 and B7-C4AQ0-C4B1-DR3.

    Conclusion: The study shows that a C4A gene deletion is the most common genetic basis for C4AQ0. It accounts for 2/3 of C4AQ0 and is found on different MHC haplotypes. 1/3 of C4AQ0 is due to other yet undefined genetic changes. The results thus demonstrate a heterogeneous genetic background for C4AQ0, giving further support for the hypothesis that C4AQ0 may be an independent risk factor for SLE.

    Keywords
    C4AQ0, C4A gene deletion, genetics, SLE, long range PCR, multiplex families
    National Category
    Immunology in the medical area
    Research subject
    Medicine
    Identifiers
    urn:nbn:se:uu:diva-107209 (URN)10.1080/03009740410011208 (DOI)15794202 (PubMedID)
    Available from: 2009-08-03 Created: 2009-07-29 Last updated: 2018-01-13Bibliographically approved
    2. Mannan-binding lectin and complement C4A in Icelandic multicase families with systemic lupus erythematosus
    Open this publication in new window or tab >>Mannan-binding lectin and complement C4A in Icelandic multicase families with systemic lupus erythematosus
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    2006 (English)In: Annals of the Rheumatic Diseases, ISSN 0003-4967, E-ISSN 1468-2060, Vol. 65, no 11, p. 1462-7Article in journal (Refereed) Published
    Abstract [en]

    Objective: To determine whether low mannan-binding lectin (MBL) and C4A null alleles (C4AQ0) are associated with systemic lupus erythematosus (SLE) in multicase families with SLE.

    Methods: Low MBL level was determined by measuring serum levels and by genotyping for mutant structural (B/C/D, designated as 0) and promoter (LX) alleles (by real-time polymerase chain reaction). C4AQ0 was detected by protein electrophoresis and corroborated with haplotype and genotype analysis. In nine Icelandic families, 24 patients with SLE were compared with 83 first-degree and 23 second-degree relatives without SLE. Twenty four unrelated family members and a population group of 330 Icelanders served as controls.

    Results: Overall, the frequency of low MBL genotypes (0/0, LX/0 and wild-type/0) tended to be higher in patients with SLE than in their first-degree and second-degree relatives (p = 0.06), but the frequency was similar in the families and in the controls (p = 0.6). The frequency of C4AQ0 was, however, increased in patients and their relatives compared with that in the controls (p = 0.04). The combination of low MBL genotypes and C4AQ0 was found more often in the patients than in their relatives (p = 0.03) and controls (p = 0.02). However, low MBL level was observed only in patients and first-degree relatives in five of the nine multicase families. In these five families, patients with SLE had low MBL genotypes more often (64%) than their first-degree (38%) and second-degree (0%) relatives (p = 0.001), and the patients with SLE also had, accordingly, lower MBL levels than their relatives (p = 0.001).

    Conclusions: These findings indicate that low MBL levels can predispose people to SLE and highlight the genetic heterogeneity of this disease.

    National Category
    Immunology in the medical area
    Research subject
    Medicine
    Identifiers
    urn:nbn:se:uu:diva-107261 (URN)10.1136/ard.2005.046086 (DOI)16439442 (PubMedID)
    Available from: 2009-08-03 Created: 2009-08-02 Last updated: 2018-01-13Bibliographically approved
    3. Lower expression levels of the programmed death 1 receptor on CD4+CD25+ T cells and correlation with the PD-1.3A genotype in patients with systemic lupus erythematosus
    Open this publication in new window or tab >>Lower expression levels of the programmed death 1 receptor on CD4+CD25+ T cells and correlation with the PD-1.3A genotype in patients with systemic lupus erythematosus
    Show others...
    2010 (English)In: Arthritis and Rheumatism, ISSN 0004-3591, E-ISSN 1529-0131, Vol. 62, no 6, p. 1702-1711Article in journal (Refereed) Published
    Abstract [en]

    OBJECTIVE.: A genetic polymorphism, PD1.3A, in the PDCD1 gene encoding the co-inhibitory immunoreceptor PD-1, has been associated with SLE. The aim of the study was to assess PD-1 receptor expression in SLE patients, relatives and controls and correlate with PD-1.3A. METHODS.: Icelandic and Swedish SLE patients, relatives and controls were studied. PBMCs were stimulated with alphaCD3/CD28 and PD-1 expression analyzed by flow cytometry. PD-1.3A/G genotyping was performed by PCR-RFLP. RESULTS: I. PD-1 expression on PBMCs was induced after stimulation, by 2.1-fold in SLE patients, 3.1-fold in relatives and 5.1-fold in controls.II. The frequency of PD-1+ cells was significantly lower in SLE patients compared to relatives and controls. PD-1 expression on PD-1+ cells was significantly lower in SLE patients and relatives.III. PD-1 expression on CD4+CD25+ T cells was significantly lower in SLE patients and relatives.IV. PD-1 expression was significantly higher on CD25(high) compared to CD25(intermediate) and (low) cells.V. PD-1 expression on CD25(high) and CD25(intermediate) cells was significantly lower in SLE patients compared to controls.VI. PD-1 was expressed on both FoxP3- and FoxP3+ cells.VII. Lower PD-1 expression was significantly correlated with the PD-1.3A/G genotype. CONCLUSION.: The study demonstrates significantly lower PD-1 receptor expression in SLE patients and relatives and a significant correlation of lower PD-1 expression with the PD-1.3A allele. We conclude that PD-1.3A may be contributory to abnormalities in PD-1 receptor expression on CD4+CD25+ T-cells in SLE, providing support for an important role for the PD-1 pathway in SLE and possibly other autoimmune diseases.

    Keywords
    SLE, PD-1.3A, self-tolerance
    National Category
    Immunology in the medical area
    Research subject
    Immunology
    Identifiers
    urn:nbn:se:uu:diva-107272 (URN)10.1002/art.27417 (DOI)000279432500020 ()20178125 (PubMedID)
    Available from: 2009-08-03 Created: 2009-08-03 Last updated: 2018-01-13Bibliographically approved
    4. Association of three systemic lupus erythematosus susceptibility factors, PD-1.3A, C4AQ0, and low levels of mannan-binding lectin, with autoimmune manifestations in Icelandic multicase systemic lupus erythematosus families
    Open this publication in new window or tab >>Association of three systemic lupus erythematosus susceptibility factors, PD-1.3A, C4AQ0, and low levels of mannan-binding lectin, with autoimmune manifestations in Icelandic multicase systemic lupus erythematosus families
    Show others...
    2008 (English)In: Arthritis and Rheumatism, ISSN 0004-3591, E-ISSN 1529-0131, Vol. 58, no 12, p. 3865-3872Article in journal (Refereed) Published
    Abstract [en]

    Objective: To study autoimmune diseases and autoantibodies in Icelandic multicase systemic lupus erythematosus (SLE) families and to determine the association of 3 SLE susceptibility factors, PD-1.3A, C4AQ0, and low levels of mannan-binding lectin (MBL), with autoimmune disease in this population.

    Methods: Eight SLE multicase families were studied, comprising a total of 124 family members (23 patients with SLE and 101 relatives). The diagnosis of an autoimmune disease was established and autoantibodies were measured in each family. In addition, PD-1.3A alleles were genotyped, and C4AQ0 allotypes were established by electrophoresis and haplotype analysis. Low levels of MBL were determined using enzyme-linked immunosorbent assay and variant-allele genotyping.

    Results: In the SLE multicase families there was a high frequency of other autoimmune diseases (32.2%) and a high frequency of autoantibodies (53.2%). Of all family members, 59.7% were determined to have SLE, other autoimmune diseases, antinuclear antibodies, and/or other autoantibodies. The families showed genetic heterogeneity for PD-1.3A, C4AQ0, and low MBL levels; the frequency of each factor ranged from 0% to 85%. The frequencies of PD-1.3A and C4AQ0 were significantly increased in patients with SLE, relatives with other autoimmune diseases, and non-autoimmune disease relatives compared with controls. In the 7 families whose members had low levels of MBL, this factor was significantly associated with SLE, but the frequency of low MBL was decreased in relatives with other autoimmune diseases as compared with non-autoimmune disease relatives and controls. There were indications of an additive effect, and 91% of patients with SLE, 78% of relatives with other autoimmune diseases, and 75% of non-autoimmune disease relatives carried at least 1 of the 3 factors.

    Conclusion: These results demonstrate a high frequency of autoimmune diseases and autoantibodies in SLE multicase families. PD-1.3A and C4AQ0 are part of a predisposing genetic background. Other genetic and/or environmental factors are necessary for disease expression, demonstrated by a high frequency of PD-1.3A and C4AQ0 in non-autoimmune disease relatives. Low MBL levels may be one such contributing factor. The results of this study provide an example of epistatic genetic effects and overlapping genetics in autoimmune diseases.

    Keywords
    SLE, PD-1, C4AQ0, low MBL, families, autoimmune disease
    National Category
    Immunology in the medical area
    Research subject
    Immunology
    Identifiers
    urn:nbn:se:uu:diva-107268 (URN)10.1002/art.24129 (DOI)000261587500025 ()19035512 (PubMedID)
    Available from: 2009-08-03 Created: 2009-08-03 Last updated: 2018-01-13Bibliographically approved
  • 24.
    Kristjánsdóttir, Helga
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Saevarsdottir, Saedis
    Gröndal, Gerdur
    Alcarcón-Riquelme, Marta E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Elendsson, Kristjan
    Valdimarsson, Helgi
    Steinsson, Kristjan
    Association of three systemic lupus erythematosus susceptibility factors, PD-1.3A, C4AQ0, and low levels of mannan-binding lectin, with autoimmune manifestations in Icelandic multicase systemic lupus erythematosus families2008In: Arthritis and Rheumatism, ISSN 0004-3591, E-ISSN 1529-0131, Vol. 58, no 12, p. 3865-3872Article in journal (Refereed)
    Abstract [en]

    Objective: To study autoimmune diseases and autoantibodies in Icelandic multicase systemic lupus erythematosus (SLE) families and to determine the association of 3 SLE susceptibility factors, PD-1.3A, C4AQ0, and low levels of mannan-binding lectin (MBL), with autoimmune disease in this population.

    Methods: Eight SLE multicase families were studied, comprising a total of 124 family members (23 patients with SLE and 101 relatives). The diagnosis of an autoimmune disease was established and autoantibodies were measured in each family. In addition, PD-1.3A alleles were genotyped, and C4AQ0 allotypes were established by electrophoresis and haplotype analysis. Low levels of MBL were determined using enzyme-linked immunosorbent assay and variant-allele genotyping.

    Results: In the SLE multicase families there was a high frequency of other autoimmune diseases (32.2%) and a high frequency of autoantibodies (53.2%). Of all family members, 59.7% were determined to have SLE, other autoimmune diseases, antinuclear antibodies, and/or other autoantibodies. The families showed genetic heterogeneity for PD-1.3A, C4AQ0, and low MBL levels; the frequency of each factor ranged from 0% to 85%. The frequencies of PD-1.3A and C4AQ0 were significantly increased in patients with SLE, relatives with other autoimmune diseases, and non-autoimmune disease relatives compared with controls. In the 7 families whose members had low levels of MBL, this factor was significantly associated with SLE, but the frequency of low MBL was decreased in relatives with other autoimmune diseases as compared with non-autoimmune disease relatives and controls. There were indications of an additive effect, and 91% of patients with SLE, 78% of relatives with other autoimmune diseases, and 75% of non-autoimmune disease relatives carried at least 1 of the 3 factors.

    Conclusion: These results demonstrate a high frequency of autoimmune diseases and autoantibodies in SLE multicase families. PD-1.3A and C4AQ0 are part of a predisposing genetic background. Other genetic and/or environmental factors are necessary for disease expression, demonstrated by a high frequency of PD-1.3A and C4AQ0 in non-autoimmune disease relatives. Low MBL levels may be one such contributing factor. The results of this study provide an example of epistatic genetic effects and overlapping genetics in autoimmune diseases.

  • 25.
    Kristjánsdóttir, Helga
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Steinsson, K.
    A study of the genetic basis of C4A protein deficiency. Detection of C4A gene deletion by long-range PCR and its associated haplotypes.2004In: Scandinavian Journal of Rheumatology, ISSN 0300-9742, E-ISSN 1502-7732, Vol. 33, no 6, p. 417-22Article in journal (Refereed)
    Abstract [en]

    Objective: Study the frequency of C4A gene deletions as the genetic basis of C4A protein deficiency (C4AQ0) and its associated haplotypes in Icelandic SLE families.

    Materials and methods: Nine multiplex SLE families were genotyped for C4A gene deletions using LR-PCR and MHC haplotypes were defined.

    Results: Of SLE patients, first- and second-degree relatives, 53,8%, 47,9% and 28,6% had C4AQ0, respectively. A C4A gene deletion is the genetic basis for C4AQ0 in 64,3% of SLE patients, 60,0% of first-degree and 50,0% of second-degree relatives.

    All individuals carrying haplotype B8-C4AQ0-C4B1-DR3 had a deletion and the deletion was also found on haplotypes B8-C4AQ0-C4B1-DR7 and B7-C4AQ0-C4B1-DR3.

    Conclusion: The study shows that a C4A gene deletion is the most common genetic basis for C4AQ0. It accounts for 2/3 of C4AQ0 and is found on different MHC haplotypes. 1/3 of C4AQ0 is due to other yet undefined genetic changes. The results thus demonstrate a heterogeneous genetic background for C4AQ0, giving further support for the hypothesis that C4AQ0 may be an independent risk factor for SLE.

  • 26.
    Kristjánsdóttir, Helga
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Steinsson, Kristjan
    Gunnarsson, Iva
    Gröndal, Gerdur
    Erlendsson, Kristjan
    Alarcón-Riquelme, Marta E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Lower expression levels of the programmed death 1 receptor on CD4+CD25+ T cells and correlation with the PD-1.3A genotype in patients with systemic lupus erythematosus2010In: Arthritis and Rheumatism, ISSN 0004-3591, E-ISSN 1529-0131, Vol. 62, no 6, p. 1702-1711Article in journal (Refereed)
    Abstract [en]

    OBJECTIVE.: A genetic polymorphism, PD1.3A, in the PDCD1 gene encoding the co-inhibitory immunoreceptor PD-1, has been associated with SLE. The aim of the study was to assess PD-1 receptor expression in SLE patients, relatives and controls and correlate with PD-1.3A. METHODS.: Icelandic and Swedish SLE patients, relatives and controls were studied. PBMCs were stimulated with alphaCD3/CD28 and PD-1 expression analyzed by flow cytometry. PD-1.3A/G genotyping was performed by PCR-RFLP. RESULTS: I. PD-1 expression on PBMCs was induced after stimulation, by 2.1-fold in SLE patients, 3.1-fold in relatives and 5.1-fold in controls.II. The frequency of PD-1+ cells was significantly lower in SLE patients compared to relatives and controls. PD-1 expression on PD-1+ cells was significantly lower in SLE patients and relatives.III. PD-1 expression on CD4+CD25+ T cells was significantly lower in SLE patients and relatives.IV. PD-1 expression was significantly higher on CD25(high) compared to CD25(intermediate) and (low) cells.V. PD-1 expression on CD25(high) and CD25(intermediate) cells was significantly lower in SLE patients compared to controls.VI. PD-1 was expressed on both FoxP3- and FoxP3+ cells.VII. Lower PD-1 expression was significantly correlated with the PD-1.3A/G genotype. CONCLUSION.: The study demonstrates significantly lower PD-1 receptor expression in SLE patients and relatives and a significant correlation of lower PD-1 expression with the PD-1.3A allele. We conclude that PD-1.3A may be contributory to abnormalities in PD-1 receptor expression on CD4+CD25+ T-cells in SLE, providing support for an important role for the PD-1 pathway in SLE and possibly other autoimmune diseases.

  • 27.
    Krook, Henrik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Hagberg, Anette
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Song, Zhenshung
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Wennberg, Lars
    Korsgren, Olle
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    A distinct Th1 immune response precedes the described Th2 response in Islet xenograft rejection2002In: Diabetes, ISSN 0012-1797, E-ISSN 1939-327X, Vol. 51, no 1, p. 79-86Article in journal (Refereed)
    Abstract [en]

    Previous studies using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) have demonstrated that islet xenograft rejection in mice is dominated by Th2-associated cytokines, i.e., interleukin (IL)-4 and IL-10. However, immunohistochemical stainings show that the morphological pattern in this model is more reminiscent of a delayed-type hypersensitivity (DTH) reaction, which is associated with a Th1 response. This study was designed to resolve the mechanisms of acute cellular xenograft rejection in rats transplanted with fetal porcine islet-like cell clusters (ICCs). Real-time quantitative RT-PCR was used to quantify the mRNA expression of cytokines in the grafts and lymph nodes, and the findings were related to the immunopathology of the rejecting grafts. By day 1, mRNA expression levels of IL-1 beta, IL-2, IL-12p40, interferon-gamma, and tumor necrosis factor-alpha were already induced in the lymph nodes. From days 3 to 12, an increasing amount of activated macrophages was seen in the grafts, whereas T- and NK-cells were fewer and mainly accumulated in the periphery of the grafts. Most of the ICCs were rejected by day 5. Transcripts of Th1-associated cytokines were dominant in both regional lymph nodes and in the grafts, with peak levels on days 3 and 5, respectively. The mRNA expression of IL-4 was increased on day 12, and it correlated with the infiltration of eosinophils and an increased level of xenoreactive IgG. The data presented indicate that an islet xenograft triggers a sequential activation of 1) a Th1-associated response characterized by graft destruction in a DTH-like reaction and then 2) a subsequent Th2-associated response characterized by increased levels of xenoreactive antibodies.

  • 28.
    Krook, Henrik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Wennberg, Lars
    Hagberg, Anette
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Song, Zhenshung
    Groth, Carl-Gustav
    Korsgren, Olle
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
    Immunosuppressive drugs in islet xenotransplantation: A tool for gaining further insights in the mechanisms of the rejection process2002In: Transplantation, ISSN 0041-1337, E-ISSN 1534-6080, Vol. 74, no 8, p. 1084-1089Article in journal (Refereed)
    Abstract [en]

    BACKGROUND:

    The aim of the present study was to examine the effect of tacrolimus (TAC) and prednisolone (PRE) in islet xenotransplantation and to use the immunosuppressive effects of these drugs and others to further characterize the mechanisms behind islet xenograft rejection.

    METHODS:

    Fetal porcine islet-like cell clusters (ICCs) were transplanted under the kidney capsule in Lewis rats. The animals were treated with TAC, cyclosporine A (CsA) plus 15-deoxyspergualin (DSG), CsA plus sirolimus (SIR) or CsA plus leflunomide (LEF), with or without the addition of PRE. Rejection was assessed by immunohistological evaluation 12 days after transplantation. In selected groups, the intragraft cytokine mRNA expression was analyzed with real-time quantitative reverse-transcriptase polymerase chain reaction (RT-PCR).

    RESULTS:

    In untreated rats, the ICC xenografts were completely rejected. Treatment with PRE alone had no, or only a marginal, protective effect. TAC alone at a dose of 1 or 0.5 mg/kg of body weight (BW) prevented xenograft rejection. The addition of PRE to TAC treatment had a paradoxical unfavorable effect. In contrast, when PRE was added to CsA-based protocols (CsA+DSG, CsA+SIR, or CsA+LEF), the immunosuppressive effect was slightly enhanced. In comparison with untreated rats, the messengers for interleukin (IL)-1beta, IL-2, IL-4, IL-10, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha were reduced in both CsA and TAC treated rats. Notably, the amount of IL-12p40 transcripts was only inhibited in rats given TAC alone, whereas this messenger was increased to approximately the same levels in untreated, CsA treated, and TAC plus PRE treated rats.

    CONCLUSIONS:

    TAC exerted a pronounced immunosuppressive effect in the pig-to-rat islet xenotransplantation model. So far, no other single drug protocol has shown a comparable efficacy. Notably, the graft protective effect of TAC was markedly abrogated when PRE was added to the treatment protocol, suggesting that TAC exerts its effect by a unique mechanism of action. In contrast with the other studied immunosuppressive regimens, treatment with TAC alone inhibited intragraft mRNA expression of all the Th1 associated cytokines, indicating that Th1 response is one important rejection mechanism that needs to be inhibited to achieve islet xenograft survival.

  • 29. Kuokkanen, S
    et al.
    Sundvall, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Terwilliger, J D
    Tienari, P J
    Wikstrom, J
    Holmdahl, Rikard
    Pettersson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Peltonen, L
    A putative vulnerability locus to multiple clerosis maps to 5p14-p12 in a region syntenic to the murine locus Eae21996In: Nature Genetics, ISSN 1061-4036, E-ISSN 1546-1718, Vol. 13, no 4, p. 477-480Article in journal (Refereed)
    Abstract [en]

    Multiple sclerosis (MS) is a chronic inflammatory disorder characterized by multifocal damage of myelin in the central nervous system (CNS). The prevalence of this putative autoimmune disease is 0.1% in individuals of northern European origin. Family, adoption and twin studies implicate genetic factors in the aetiology. MS is widely speculated to be a multifactorial disorder with a complex mode of inheritance. Despite many studies of candidate genes, only an association with HLA-DR2-DQ6 has been generally detected, and the number of susceptibility genes remains unknown. The chronic variant of experimental allergic encephalomyelitis (EAE), a T-cell mediated autoimmune disease in rodents, represents a relevant animal model for MS given the chronic relapsing disease course and inflammatory changes of CNS observed in these demyelinating disorders. Susceptibility to EAE is also influenced by the major histocompatibility complex (MHC). Human syntenic regions to murine loci predisposing to EAE were tested as candidate regions for genetic susceptibility of MS. Three chromosomal regions (1p22-q23, 5p14-p12 and Xq13.2-q22) were screened in 21 Finnish multiplex MS families most originating from a high risk region in western Finland. Several markers yielded positive lod scores on 5p14-p12, syntenic to the murine locus Eae2. Our data provide evidence for a predisposing locus for MS on 5p14-p12.

  • 30.
    Lagerstedt, Kristina
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Karsten, Stanislav
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Carlberg, Britt-Marie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Kleijer, Wim J
    Tönnesen, Tönne
    Pettersson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Bondeson, Marie-Louise
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Double-strand breaks may initiate the inversion mutation causing the Hunter syndrome1997In: Human Molecular Genetics, ISSN 0964-6906, E-ISSN 1460-2083, Vol. 6, no 4, p. 627-633Article in journal (Refereed)
    Abstract [en]

    We have previously shown that patients with the Hunter syndrome frequently have suffered from a recombination event between the IDS gene and its putative pseudogene, IDS-2, resulting in an inversion of the intervening DNA. The inversion, which might be the consequence of an intrachromosomal mispairing, is caused by homologous recombination between sequences located in intron 7 of the IDS gene and sequences located distal of exon 3 in IDS-2. In order to gain insight into the mechanisms causing the inversion, we have isolated both inversion junctions in six unrelated patients. DNA sequence analysis of the junctions showed that all recombinations have taken place within a 1 kb region where the sequence identity is >98%. An interesting finding was the identification of regions with alternating IDS gene and IDS-2 sequences present at one inversion junction, suggesting that the recombination event has been initiated by a double-strand break in intron 7 of the IDS gene. The results from this study suggest that homologous recombination in man could be explained by mechanisms similar to those described for Saccharomyces cerevisiae. The results also have practical implications for diagnosis of patients with the Hunter syndrome.

  • 31.
    Lagerström-Fermér, Maria
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Nilsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Backman, B
    Salido, E
    Shapiro, L
    Pettersson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Amelogenin signal peptide mutation: correlation between mutations in the amelogenin gene (AMGX) and manifestations of X-linked amelogenesis imperfecta1995In: Genomics, ISSN 0888-7543, E-ISSN 1089-8646, Vol. 26, no 1, p. 159-162Article in journal (Refereed)
    Abstract [en]

    Formation of tooth enamel is a poorly understood biological process. In this study we describe a 9-bp deletion in exon 2 of the amelogenin gene (AMGX) causing X-linked hypoplastic amelogenesis imperfecta, a disease characterized by defective enamel. The mutation results in the loss of 3 amino acids and exchange of 1 in the signal peptide of the amelogenin protein. This deletion in the signal peptide probably interferes with translocation of the amelogenin protein during synthesis, resulting in the thin enamel observed in affected members of the family. We compare this mutation to a previously reported mutation in the amelogenin gene that causes a different disease phenotype. The study illustrates that molecular analysis can help explain the various manifestations of a tooth disorder and thereby provide insights into the mechanisms of tooth enamel formation.

  • 32.
    Lagerström-Fermér, Maria
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Sundvall, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Johnsen, Elsy
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Warne, GL
    Forrest, SM
    Zajac, JD
    Richards, A
    Ravine, D
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Pettersson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    X-linked recessive panhypopituitarism associated with a regional duplication in Xq25-q261997In: American Journal of Human Genetics, ISSN 0002-9297, E-ISSN 1537-6605, Vol. 60, no 4, p. 910-916Article in journal (Refereed)
    Abstract [en]

    We present a linkage analysis and a clinical update on a previously reported family with X-linked recessive panhypopituitarism, now in its fourth generation. Affected members exhibit variable degrees of hypopituitarism and mental retardation. The markers DXS737 and DXS1187 in the q25-q26 region of the X chromosome showed evidence for linkage with a peak LOD score (Zmax) of 4.12 at zero recombination fraction (theta(max) = 0). An apparent extra copy of the marker DXS102, observed in the region of the disease gene in affected males and heterozygous carrier females, suggests that a segment including this marker is duplicated. The gene causing this disorder appears to code for a dosage-sensitive protein central to development of the pituitary.

  • 33. Larsson, P A
    et al.
    Hirsch, Jan M
    Department of Oral Surgery, University of Göteborg, S-413 46 Göteborg, Sweden.
    Gronowitz, J S
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Vahlne, A
    Inhibition of herpes simplex virus replication and protein synthesis by non-smoked tobacco, tobacco alkaloids and nitrosamines1992In: Archives of Oral Biology, ISSN 0003-9969, E-ISSN 1879-1506, Vol. 37, no 11, p. 969-978Article in journal (Refereed)
    Abstract [en]

    Inhibitory effects of snuff extract and the tobacco chemicals nicotine, anabasine, diethyl-N-nitrosamine (DEN), and the tobacco-specific nitrosamines (TSNA), N-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) on herpes simplex virus type 1 (HSV-1) replication in vitro and on HSV-1 protein synthesis in infected cells were analysed. Snuff extract and nicotine caused a significant reduction of HSV-1 attachment to cell membranes whereas anabasine, DEN, NNN and NNK did not affect adsorption of HSV-1. Virus production assays in the presence of snuff added after virus adsorption resulted in a significantly reduced production of virus at low multiplicities of infection (MOI), but at high MOI the inhibitory effect of snuff extract was less pronounced. DEN, NNN and NNK only affected virus production at toxic concentrations. Nicotine and anabasine reduced virus production in non-toxic doses but not at the concentrations present in snuff extract. In HSV-infected cells exposed to snuff extract, the immediate early (α-) infected cell proteins (ICPs) 4 and 27 (as well as the early (β-) ICPs 6 and 8) were markedly increased, whereas the late (γ-) ICPs 5, 11 and 29 were reduced. Nicotine had a less pronounced stimulating effect on the production of α-proteins but no detectable effect on production of β- or -γ-proteins. Anabasine, DEN, NNN and NNK did not affect HSV protein synthesis at non-toxic concentrations. Synthesis of thymidine kinase and DNA polymerase was significantly reduced by snuff extract. Also nicotine and anabasine affected thymidine kinase and DNA polymerase but only at toxic concentrations. The production of the cellular protein actin, which almost disappears a few hours after HSV-1 infection, remained at a significant level in HSV-infected cells exposed to snuff. Thus snuff extract blocks the replicative cycle of HSV at an early stage, which results in an increased production of α-proteins in the infected cells and in prolonged maintenance of cellular functions. This may be of importance for HSV-induced transformation and the development of HSV-associated tumours.

  • 34.
    Lennerstrand, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Rytting, A S
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Örvell, C
    Gronowitz, J S
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Källander, C F
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    A Method for Combined Immunoaffinity Purification and Assay of HIV-1 Reverse Transcriptase Activity Useful for Crude Samples1996In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 235, no 2, p. 141-152Article in journal (Refereed)
    Abstract [en]

    Detection of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity in crude specimens was greatly enhanced using a novel capture RT assay. Eighteen different monoclonal antibodies (Mabs) raised against purified HIV-1 RT were tested for their ability to bind to HIV-1 RT without affecting its activity. The anti-HIV-1 RT Mabs were immobilized on plastic macrobeads and used as solid carriers in the capture RT assay. The assay system first involved RT's adherence to the immobilized Mabs. Nonspecific enzymes and other impurities were removed by a simple wash after which the RT reaction mixture was added. Substrate and product were finally separated by a wash of the beads. Practically all radioactivity incorporated into DNA (>98%) was recovered on the bead. The Michaelis-Menten constants and the saturation velocity values for the nucleotide substrate were similar for free and immobilized RT. The reaction mechanism for the immobilized RT is discussed. When comparing the function of this assay with more conventional soluble RT assays for samples consisting of recombinant HIV-1 RT mixed with an extract of peripheral blood lymphocytes (PBL), an almost 100-fold higher sensitivity was found. The capture RT assay had the capacity to recover approximately 80% of the RT activity added to an extract of 1 x 10(7) PBL cells/ ml. A strong correlation (r = 0.947) between the results obtained with this assay and a HIV-1 p24 enzyme-linked immunosorbent assay was found, when samples from a collection of 16 HIV strains propagated in cell culture were analyzed.

  • 35. Lindblad, K
    et al.
    Nylander, PO
    Zander, C
    Yuan, QP
    Ståhle, L
    Engström, C
    Balciuniene, J
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Pettersson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Breschel, T
    McInnis, M
    Ross, CA
    Adolfsson, R
    Schalling, M
    Two commonly expanded CAG/CTG repeat loci: involvement in affectivedisorders?1998In: Molecular Psychiatry, ISSN 1359-4184, E-ISSN 1476-5578, Vol. 3, no 5, p. 405-410Article in journal (Refereed)
    Abstract [en]

    An association between bipolar affective disorder and CAG/CTG trinucleotide repeat expansions (TRE) has previously been detected using the repeat expansion detection (RED) method. Here we report that 89% of RED products (CAG/CTG repeats) > 120 nt (n = 202) detected in affective disorder patients as well as unaffected family members and controls correlate with expansions at two repeat loci, ERDA1 on chromosome 17q21.3 and CTG18.1 on 18q21.1. In a set of patients and controls in which we had previously found a significant difference in RED size distribution, the frequency of expansions at the CTG18.1 locus was 13% in bipolar patients (n = 60) and 5% in controls (n = 114) (P < 0.07) with a significantly different size distribution (P < 0.03). A second set of patients were ascertained from 14 affective disorder families showing anticipation. Twelve of the families had members with RED products > 120 nt. The RED product distribution was significantly different (P < 0.0007) between affected (n = 53) and unaffected (n = 123) offspring. Using PCR, a higher frequency (P < 0.04) of CTG18.1 expansions as well as a different (P < 0.02) repeat size distribution was seen between affected and unaffected offspring. In addition, a negative correlation between RED product size and the age-of-onset could be seen in affected offspring (rs = -0.3, P = 0.05, n = 43). This effect was due to an earlier onset in individuals with long CTG18.1 expansions. No difference in ERDA1 expansion frequency was seen either between bipolar patients (35%, n = 60) and matched controls (29%, n = 114), or between affected and unaffected offspring in the families. We conclude that expanded alleles at the CTG18.1 locus confers an odds ratio of 2.6-2.8 and may thus act as a vulnerability factor for affective disorder, while the ERDA1 locus seems unrelated to disease.

  • 36.
    Malmgren, Helena
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Carlberg, Britt-Marie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Pettersson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Bondeson, Marie-Louise
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Identification of an alternative transcript fromthe human iduronate-2-sulfatase (IDS) gene1995In: Genomics, ISSN 0888-7543, E-ISSN 1089-8646, Vol. 29, no 1, p. 291-293Article in journal (Refereed)
    Abstract [en]

    Iduronate-2-sulfatase (IDS) is involved in the degradation of heparan sulfate and dermatan sulfate in the lysosomes, and a deficiency in this enzyme results in Hunter syndrome. A 2.3-kb cDNA clone that contains the entire coding sequence of IDS has previously been reported. Here we describe the identification of a 1.4-kb transcript that may encode an IDS-like enzyme. The predicted protein is identical to the previously described enzyme, except for the absence of the 207-amino-acid COOH-terminal domain, which is replaced by 7 amino-acids. Our data suggest that there might exist an additional form of the IDS enzyme in humans. The results from this study may have implications for the pathogenesis of the Hunter syndrome.

  • 37. Porcel, Betina
    et al.
    Bontempi, Esteban
    Henriksson, Jan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Rydåker, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Åslund, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Segura, EL
    Pettersson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Ruiz, A M
    Trypanosoma rangeli and Trypanosoma cruzi: molecular characterization of genes encoding putative calcium-binding proteins, highly conserved in typanosomatids1996In: Experimental parasitology, ISSN 0014-4894, E-ISSN 1090-2449, Vol. 84, no 3, p. 387-399Article in journal (Refereed)
    Abstract [en]

    Genes encoding a 29-kDa flagellar calcium-binding protein (F29) in Trypanosoma cruzi, strongly homologous to EF-hand calcium-binding protein-encoding genes previously reported in this parasite, were isolated by immunoscreening. F29 is encoded by a number of very similar genes, highly conserved among different T. cruzi isolates. The genes are located on a pair of homologous chromosomes, arranged in one or two clusters of tandem repeats. PCR amplification of Trypanosoma rangeli genomic DNA, using primers derived from the T. cruzi F29 sequence made it possible to isolate the homologous gene in T. rangeli, encoding a 23-kDa protein called TrCaBP. Gene sequence comparisons showed homology to EF-hand calcium-binding proteins from T. cruzi (82.8%), Trypanosoma brucei brucei (60.2%), and Entamoeba histolytica (28.4%). Northern blot analysis revealed that the TrCaBP gene is expressed in T. rangeli as a polyadenylated transcript. The TrCaBP-encoding genes are present in at least 20 copies per cell, organized in tandem arrays, on large T. rangeli chromosomes in some isolates and on two smaller ones in others. This gene, however, seems to be absent from Leishmania.

  • 38. Sabaj, V
    et al.
    Åslund, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Pettersson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Galanti, N
    Histone genes expression during the cell cycle in Trypanosoma cruzi2001In: Journal of Cellular Biochemistry, ISSN 0730-2312, E-ISSN 1097-4644, Vol. 80, no 4, p. 617-624Article in journal (Refereed)
    Abstract [en]

    Histones, the basic proteins which compact DNA into the nucleosomal and solenoidal fibers are synthesized in correlation with DNA replication during the S-phase of the cell cycle. This behavior is controlled both at transcriptional and postranscriptional levels in higher eukaryotes and yeasts. We have found that histone synthesis in synchronized trypanosomes is controlled by fluctuations on the levels of their mRNAs. Though we cannot preclude the existence of a transcriptional regulatory mechanism, our results point to the participation of changes in the stability of histone mRNAs as a regulatory mechanism of their levels during the cell cycle in Trypanosoma. We have also found a postranscriptional regulatory mechanism which could be acting at the translational level. These results show both similarities and differences between Trypanosoma and higher eukaryotes regarding the expression of their histone genes.

  • 39.
    Saetre, Peter
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Evolutionary Biology.
    Lindberg, Julia
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Evolutionary Biology.
    Leonard, Jennifer A
    Olsson, Kerstin
    Petterson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Ellegren, Hans
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Evolutionary Biology.
    Bergström, Tomas F
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Vilà, Carles
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Evolutionary Biology.
    Jazin, Elena
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Evolutionary Biology.
    From wild wolf to domestic dog: gene expression changes in the brain2004In: Brain Research. Molecular Brain Research, ISSN 0169-328X, E-ISSN 1872-6941, Vol. 126, no 2, p. 198-206Article in journal (Refereed)
    Abstract [en]

    Despite the relatively recent divergence time between domestic dogs (Canis familiaris) and gray wolves (Canis lupus), the two species show remarkable behavioral differences. Since dogs and wolves are nearly identical at the level of DNA sequence, we hypothesize that the two species may differ in patterns of gene expression.

    We compare gene expression patterns in dogs, wolves and a close relative, the coyote (Canis latrans), in three parts of the brain: hypothalamus, amygdala and frontal cortex, with microarray technology. Additionally, we identify genes with region-specific expression patterns in all three species. Among the wild canids, the hypothalamus has a highly conserved expression profile. This contrasts with a marked divergence in domestic dogs. Real-time PCR experiments confirm the altered expression of two neuropeptides, CALCB and NPY. Our results suggest that strong selection on dogs for behavior during domestication may have resulted in modifications of mRNA expression patterns in a few hypothalamic genes with multiple functions. This study indicates that rapid changes in brain gene expression may not be exclusive to the development of human brains. Instead, they may provide a common mechanism for rapid adaptive changes during speciation, particularly in cases that present strong selective pressures on behavioral characters.

  • 40. Saevarsdottir, S.
    et al.
    Kristjánsdóttir, Helga
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Gröndal, G.
    Vikingsdottir, T.
    Steinsson, K.
    Valdimarsson, H.
    Mannan-binding lectin and complement C4A in Icelandic multicase families with systemic lupus erythematosus2006In: Annals of the Rheumatic Diseases, ISSN 0003-4967, E-ISSN 1468-2060, Vol. 65, no 11, p. 1462-7Article in journal (Refereed)
    Abstract [en]

    Objective: To determine whether low mannan-binding lectin (MBL) and C4A null alleles (C4AQ0) are associated with systemic lupus erythematosus (SLE) in multicase families with SLE.

    Methods: Low MBL level was determined by measuring serum levels and by genotyping for mutant structural (B/C/D, designated as 0) and promoter (LX) alleles (by real-time polymerase chain reaction). C4AQ0 was detected by protein electrophoresis and corroborated with haplotype and genotype analysis. In nine Icelandic families, 24 patients with SLE were compared with 83 first-degree and 23 second-degree relatives without SLE. Twenty four unrelated family members and a population group of 330 Icelanders served as controls.

    Results: Overall, the frequency of low MBL genotypes (0/0, LX/0 and wild-type/0) tended to be higher in patients with SLE than in their first-degree and second-degree relatives (p = 0.06), but the frequency was similar in the families and in the controls (p = 0.6). The frequency of C4AQ0 was, however, increased in patients and their relatives compared with that in the controls (p = 0.04). The combination of low MBL genotypes and C4AQ0 was found more often in the patients than in their relatives (p = 0.03) and controls (p = 0.02). However, low MBL level was observed only in patients and first-degree relatives in five of the nine multicase families. In these five families, patients with SLE had low MBL genotypes more often (64%) than their first-degree (38%) and second-degree (0%) relatives (p = 0.001), and the patients with SLE also had, accordingly, lower MBL levels than their relatives (p = 0.001).

    Conclusions: These findings indicate that low MBL levels can predispose people to SLE and highlight the genetic heterogeneity of this disease.

  • 41.
    Sundvall, Mats
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Jirholt, J
    Yang, Hai-Tao
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Jansson, L
    Engström, Åke
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Pettersson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Holmdahl, Rikard
    Department ofMedical Inflammation Research, Lund University, Lund, Sweden.
    Identification of murine loci associated with susceptibility to chronic experimental autoimmune encephalomyelitis1995In: Nature Genetics, ISSN 1061-4036, E-ISSN 1546-1718, Vol. 10, no 3, p. 313-317Article in journal (Refereed)
    Abstract [en]

    B10.RIII mice develop chronic and relapsing experimental autoimmune encephalomyelitis (EAE) after immunization with the myelin basic protein (MBP) peptide 89−101. The disease is associated with the major histocompatibility complex (MHC) (eae1). We have now investigated the importance of non−MHC regions for the EAE susceptibility in a cross between RIIIS/J and B10.RIII mice which share the MHC region but differ in disease susceptibility. Linkage analysis using microsatellite markers spanning the genome identified a region (eae2) on chromosome 15 which showed linkage to disease (P=0.0002). Our data also suggest linkage to a second region (eae3) on chromosome 3 (P=0.0024), and provide evidence for locus interactions between eae2 and eae3. These results provide clues to the genetic basis of multiple sclerosis.

  • 42. Syvänen, Ann-Christine
    et al.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Detection of point mutations by solid-phase methods1994In: Human Mutation, ISSN 1059-7794, E-ISSN 1098-1004, Vol. 3, no 3, p. 172-179Article in journal (Refereed)
    Abstract [en]

    Several techniques exist that permit the efficient distinction among characterized DNA sequence variants. In this review we discuss a number of such analytic procedures. These techniques all take advantage of a variety solid supports to prepare and analyze reaction products. The described diagnostic principles are now being applied for the development of miniaturized assay formats, suitable for automated detection of large sets of sequences in clinical samples.

  • 43.
    Thuresson, Ann-Charlotte
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Genetics.
    Aström, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Genetics.
    Aström, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Genetics.
    Grönvik, Kjell Olov
    The National Veterinary Institute, Section of Immunology and Cell Culture, Biomedical Center, Uppsala.
    Virtanen, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Multiple forms of poly(A) polymerases in human cells1994In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 91, no 3, p. 979-983Article in journal (Refereed)
    Abstract [en]

    We have cloned human poly(A) polymerase (PAP) mRNA as cDNA in Escherichia coli. The primary structure of the mRNA was determined and compared to the bovine PAP mRNA sequence. The two sequences were 97% identical at the nucleotide level, which translated into 99% similarity at the amino acid level. Polypeptides representing recombinant PAP were expressed in E. coli, purified, and used as antigens to generate monoclonal antibodies. Western blot analysis using these monoclonal antibodies as probes revealed three PAPs, having estimated molecular masses of 90, 100, and 106 kDa in HeLa cell extracts. Fractionation of HeLa cells showed that the 90-kDa polypeptide was nuclear while the 100- and 106-kDa species were present in both nuclear and cytoplasmic fractions. The 106-kDa PAP was most likely a phosphorylated derivative of the 100-kDa species. PAP activity was recovered in vitro by using purified recombinant human PAP. Subsequent mutational analysis revealed that both the N- and C-terminal regions of PAP were important for activity and suggested that cleavage and polyadenylylation specificity factor (CPSF) interacted with the C-terminal region of PAP. Interestingly, tentative phosphorylation sites have been identified in this region, suggesting that phosphorylation/dephosphorylation may regulate the interaction between the two polyadenylylation factors PAP and CPSF.

  • 44. Timms, Kirsten M
    et al.
    Bondesson, Marie-Louise
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Ansari-Lari, Ali M
    Lagerstedt, Kristina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Muzny, Donna M
    Dugan-Rocha, Shannon P
    Nelson, David L
    Pettersson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Gibbs, Richard
    Molecular and phenotypic variation in patients with severe Hunter syndrome1997In: Human Molecular Genetics, ISSN 0964-6906, E-ISSN 1460-2083, Vol. 6, no 3, p. 479-486Article in journal (Refereed)
    Abstract [en]

    Severe Hunter syndrome is a fatal X-linked lysosomal storage disorder caused by iduronate-2-sulphatase (IDS) deficiency. Patients with complete deletion of the IDS locus often have atypical phenotypes including ptosis, obstructive sleep apnoea, and the occurrence of seizures. We have used genomic DNA sequencing to identify several new genes in the IDS region. DNA deletion patients with atypical symptoms have been analysed to determine whether these atypical symptoms could be due to involvement of these other loci. The occurrence of seizures in two individuals correlated with a deletion extending proximal of IDS, up to and including part of the FMR2 locus. Other (non-seizure) symptoms were associated with distal deletions. In addition, a group of patients with no variant symptoms, and a characteristic rearrangement involving a recombination between the IDS gene and an adjacent IDS pseudogene (IDS psi), showed normal expression of loci distal to IDS. Together, these results identify FMR2 as a candidate gene for seizures, when mutated along with IDS.

  • 45.
    Vahlquist, Anders
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Dermatology and Venereology.
    Bygum, Anette
    Gånemo, Agneta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Dermatology and Venereology.
    Virtanen, Marie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Dermatology and Venereology.
    Hellström-Pigg, Maritta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Strauss, Gitte
    Brandrup, Flemming
    Fischer, Judith
    Genotypic and clinical spectrum of self-improving collodion ichthyosis: ALOX12B, ALOXE3, and TGM1 mutations in Scandinavian patients2010In: Journal of Investigative Dermatology, ISSN 0022-202X, E-ISSN 1523-1747, Vol. 130, no 2, p. 438-443Article in journal (Refereed)
    Abstract [en]

    Infants born with autosomal recessive congenital ichthyosis (ARCI) are often encapsulated in a collodion membrane, which shows a lamellar or erythrodermic type of ichthyosis upon shedding. However, some babies show a nearly normal underlying skin after several weeks, a phenotype called "self-healing collodion baby" (SHCB). Mutations in two genes, TGM1 and ALOX12B, have previously been implicated in the etiology of SHCB, but the full genotypic spectrum remains to be determined. DNA sequencing in 11 Swedish and 4 Danish SHCB patients showed ALOX12B mutations in eight cases, ALOXE3 mutations in three cases, and TGM1 mutations in one case. In three patients, we could not find mutations in any of the known ARCI genes. In all cases, a spontaneous shedding of the collodion membrane occurred 2-4 weeks after birth. When re-examined at 2-37 years of age, the patients showed skin xerosis, a mild or focal scaling, palmar hyperlinearity with keratoderma, and a frequent appearance of red cheeks and anhidrosis. Thus, we propose replacing SHCB with the term "self-improving collodion ichthyosis" (SICI). In conclusion, ALOX12B mutations are the leading cause of SICI in Scandinavia, followed by ALOXE3 mutations, which have not been previously associated with this variant of ARCI.

  • 46. van Holst Pellekaan, Sheila M.
    et al.
    Ingman, Max
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Roberts-Thomson, June
    Harding, Rosalind M.
    Mitochondrial genomics identifies major haplogroups in Aboriginal Australians2006In: American Journal of Physical Anthropology, ISSN 0002-9483, E-ISSN 1096-8644, Vol. 131, no 2, p. 282-294Article in journal (Refereed)
    Abstract [en]

    We classified diversity in eight new complete mitochondrial genome sequences and 41 partial sequences from living Aboriginal Australians into five haplogroups. Haplogroup AuB belongs to global lineage M, and AuA, AuC, AuD, and AuE to N. Within N, we recognize subdivisions, assigning AuA to haplogroup S, AuD to haplogroup 0, AuC to P4, and AuE to P8. On available evidence, (S)AuA and (AuB)-Au-M are widespread in Australia. (AuC)-Au-P4 is found in the Riverine region of western New South Wales, and was identified by others in northern Australia. (AuD)-Au-O and (AuE)-Au-P8 were clearly identified only from central Australia. Our eight Australian full mt genome sequences, combined with 20 others (Ingman and Gyllensten [2003] Genome Res. 13:1600-1606) and compared with full mt genome sequences from regions to the north that include Papua New Guinea, Malaya, and Andaman and Nicobar Islands, show that ancestral connections between regions are deep and limited to clustering at the level of the N and M macro-haplogroups. The Australian-specific distribution of the five haplogroups identified indicates genetic isolation over a long period. Ancestral connections within Australia are deeper than those reflected by known linguistic or culturally based affinities. Applying a coalescence analysis to a gene tree for the coding regions of the eight genomic sequences, we made estimates of time depth that support a continuity of presence for the descendants of a founding population already established by 40,000 years ago.

  • 47.
    Wadelius, Claes
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Integrating genome and epigenome in human disease2009In: Epigenomics / [ed] Anne Ferguson-Smith, John Greally & Robert Martiniensen, Dordrecht: Springer Science+Business Media B.V., 2009, p. 343-368Chapter in book (Other academic)
    Abstract [en]

    Transcription factors (TFs) and the core components of the epigenome, DNA methylation and histone modifications interact in the epigenetic machinery. Expression of key three-four selected transcription factors can dedifferentiate differentiated cells into induced pluripotent stem cells with a totally distinct epigenome. Out of the around 2,000 nuclear proteins, around 350 are known to cause disease when mutated. The conditions range from rare syndromes to common diseases. Mutations are found in all components of the epigenetic machinery, e.g. proteins mediating DNA methylation and those binding to methylated cytosine, histone proteins and enzymes adding or removing modifications to histone tails, those binding to the modifications as well as proteins in the basal transcription machinery. Sequence specific transcription factors are the most abundant nuclear proteins and mutations in them cause a wide range of diseases affecting different organs in the developing or mature organism. Even if a mutated protein is present in most or all cell-types, a specific disease is often found only in one or a few types. The reason for this is unclear and an interesting topic for future research. One hypothesis is that the disease is caused by the mutated protein interacting with other tissue restricted proteins. Technical breakthroughs like chromatin immunoprecipitation and analysis on next generation sequencers (ChIP-seq) mean that these processes can be systematically studied.

     

  • 48.
    Wallerman, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Genome-Wide Studies of Transcriptional Regulation in Mammalian Cells2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The key to the complexity of higher organisms lies not in the number of protein coding genes they carry, but rather in the intrinsic complexity of the gene regulatory networks. The major effectors of transcriptional regulation are proteins called transcription factors, and in this thesis four papers describing genome-wide studies of seven such factors are presented, together with studies on components of the chromatin and transcriptome.

    In Paper I, we optimized a large-scale in vivo method, ChIP-chip, to study protein – DNA interactions using microarrays. The metabolic-disease related transcription factors USF1, HNF4a and FOXA2 were studied in 1 % of the genome, and a surprising number of binding sites were found, mostly far from annotated genes.

    In Paper II, a novel sequencing based method, ChIP-seq, was applied to FOXA2, HNF4a and GABPa, allowing a true genome-wide view of binding sites. A large overlap between the datasets were seen, and molecular interactions were verified in vivo. Using a ChIP-seq specific motif discovery method, we identified both the expected motifs and several for co-localized transcription factors.

    In Paper III, we identified and studied a novel transcription factor, ZBED6, using the ChIP-seq method. Here, we went from one known binding site to several hundred sites throughout the mouse genome. Finally, in Paper IV, we studied the chromatin landscape by deep sequencing of nucleosomal DNA, and further used RNA-sequencing to quantify expression levels, and extended the knowledge about the binding profiles for the transcription factors NFY and TCF7L2.

    List of papers
    1. Binding sites for metabolic disease related transcription factors inferred at base pair resolution by chromatin immunoprecipitation and genomic microarrays
    Open this publication in new window or tab >>Binding sites for metabolic disease related transcription factors inferred at base pair resolution by chromatin immunoprecipitation and genomic microarrays
    Show others...
    2005 (English)In: Human Molecular Genetics, ISSN 0964-6906, E-ISSN 1460-2083, Vol. 14, no 22, p. 3435-3447Article in journal (Refereed) Published
    Abstract [en]

    We present a detailed in vivo characterization of hepatocyte transcriptional regulation in HepG2 cells, using chromatin immunoprecipitation and detection on PCR fragment-based genomic tiling path arrays covering the encyclopedia of DNA element (ENCODE) regions. Our data suggest that HNF-4α and HNF-3β, which were commonly bound to distal regulatory elements, may cooperate in the regulation of a large fraction of the liver transcriptome and that both HNF-4α and USF1 may promote H3 acetylation to many of their targets. Importantly, bioinformatic analysis of the sequences bound by each transcription factor (TF) shows an over-representation of motifs highly similar to the in vitro established consensus sequences. On the basis of these data, we have inferred tentative binding sites at base pair resolution. Some of these sites have been previously found by in vitro analysis and some were verified in vitro in this study. Our data suggests that a similar approach could be used for the in vivo characterization of all predicted/uncharacterized TF and that the analysis could be scaled to the whole genome.

    Keywords
    Base Pairing/*genetics, Binding Sites/genetics, Cell Line; Tumor, Chromatin/*metabolism, Chromatin Immunoprecipitation/methods, Consensus Sequence, Genome; Human, Hepatocyte Nuclear Factor 3-beta/physiology, Hepatocyte Nuclear Factor 4/physiology, Hepatocytes/metabolism, Histones/metabolism, Humans, Metabolic Diseases/*metabolism, Oligonucleotide Array Sequence Analysis/methods, Promoter Regions (Genetics), Research Support; N.I.H.; Extramural, Research Support; Non-U.S. Gov't, Sequence Analysis; DNA, Transcription Factors/genetics/*metabolism, Upstream Stimulatory Factors/metabolism
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-80603 (URN)10.1093/hmg/ddi378 (DOI)16221759 (PubMedID)
    Available from: 2006-05-19 Created: 2006-05-19 Last updated: 2017-12-14Bibliographically approved
    2. Molecular interactions between HNF4a, FOXA2 and GABP identified at regulatory DNA elements through ChIP-sequencing
    Open this publication in new window or tab >>Molecular interactions between HNF4a, FOXA2 and GABP identified at regulatory DNA elements through ChIP-sequencing
    Show others...
    2009 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 37, no 22, p. 7498-7508Article in journal (Refereed) Published
    Abstract [en]

    Gene expression is regulated by combinations of transcription factors, which can be mapped to regulatory elements on a genome-wide scale using ChIP experiments. In a previous ChIP-chip study of USF1 and USF2 we found evidence also of binding of GABP, FOXA2 and HNF4a within the enriched regions. Here, we have applied ChIP-seq for these transcription factors and identified 3064 peaks of enrichment for GABP, 7266 for FOXA2 and 18783 for HNF4a. Distal elements with USF2 signal was frequently bound also by HNF4a and FOXA2. GABP peaks were found at transcription start sites, whereas 94% of FOXA2 and 90% of HNF4a peaks were located at other positions. We developed a method to accurately define TFBS within peaks, and found the predicted sites to have an elevated conservation level compared to peak centers; however the majority of bindings were not evolutionary conserved. An interaction between HNF4a and GABP was seen at TSS, with one-third of the HNF4a positive promoters being bound also by GABP, and this interaction was verified by co-immunoprecipitations.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-121011 (URN)10.1093/nar/gkp823 (DOI)000272935000022 ()19822575 (PubMedID)
    Available from: 2010-03-18 Created: 2010-03-18 Last updated: 2017-12-12Bibliographically approved
    3. ZBED6, a novel transcription factor derived from a domesticated DNA transposon regulates IGF2 expression and muscle growth
    Open this publication in new window or tab >>ZBED6, a novel transcription factor derived from a domesticated DNA transposon regulates IGF2 expression and muscle growth
    Show others...
    2009 (English)In: PLoS biology, ISSN 1544-9173, E-ISSN 1545-7885, Vol. 7, no 12, p. e1000256-Article in journal (Refereed) Published
    Abstract [en]

    A single nucleotide substitution in intron 3 of IGF2 in pigs abrogates a binding site for a repressor and leads to a 3-fold up-regulation of IGF2 in skeletal muscle. The mutation has major effects on muscle growth, size of the heart, and fat deposition. Here, we have identified the repressor and find that the protein, named ZBED6, is previously unknown, specific for placental mammals, and derived from an exapted DNA transposon. Silencing of Zbed6 in mouse C2C12 myoblasts affected Igf2 expression, cell proliferation, wound healing, and myotube formation. Chromatin immunoprecipitation (ChIP) sequencing using C2C12 cells identified about 2,500 ZBED6 binding sites in the genome, and the deduced consensus motif gave a perfect match with the established binding site in Igf2. Genes associated with ZBED6 binding sites showed a highly significant enrichment for certain Gene Ontology classifications, including development and transcriptional regulation. The phenotypic effects in mutant pigs and ZBED6-silenced C2C12 myoblasts, the extreme sequence conservation, its nucleolar localization, the broad tissue distribution, and the many target genes with essential biological functions suggest that ZBED6 is an important transcription factor in placental mammals, affecting development, cell proliferation, and growth.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-119579 (URN)10.1371/journal.pbio.1000256 (DOI)000273060500005 ()20016685 (PubMedID)
    Available from: 2010-02-26 Created: 2010-02-26 Last updated: 2017-12-12Bibliographically approved
    4. Nucleosome landscape in HepG2 cells in relation to NFY, HNF4a and FOXA2 binding sites
    Open this publication in new window or tab >>Nucleosome landscape in HepG2 cells in relation to NFY, HNF4a and FOXA2 binding sites
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    Nucleosomes are the building blocks that compact the DNA in the nucleus, thereby regulating its accessibility. Here we report a genome-wide nucleosome positioning analysis on the HepG2 cell line, based on deep sequencing of mononucleosomal DNA. In concordance with other studies, we found nucleosomes to be well positioned at the TSS, with a nucleosome free region in the proximal promoter. Focusing on the importance of nucleosomal positioning at distal elements we found that TFBS sites often are flanked by well-positioning nucleosomes at a distance that indicate that the TF complexes replaces the nucleosome, and we also find that some transposable elements have distinct nucleosomal signatures. To build on previous regulatory data for HepG2 cells we also produced ChIP-seq reads for the transcription factors NF-Y and TCF7L2 and correlate this to the HepG2 transcriptome as defined by RNA-seq and Pol-II ChIP-seq. NF-Y was found primarily at promoters, contrary to what has been suggested from previous studies, and binds genes involved in cell cycle and chromatin organization.

    Keywords
    ChIP-seq, NFY, Pol-II, TCF7L2, nucleosome positioning
    National Category
    Medical Genetics
    Research subject
    Medical Genetics
    Identifiers
    urn:nbn:se:uu:diva-132879 (URN)
    Available from: 2010-10-28 Created: 2010-10-28 Last updated: 2018-01-12Bibliographically approved
  • 49. Yuan, Q P
    et al.
    Lindblad-Toh, Kerstin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Zander, Cecilia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Burgess, C
    Durr, A
    Schalling, M
    A cloning strategy for identification of genes containing trinucleotide repeat expansions2001In: International Journal of Molecular Medicine, ISSN 1107-3756, E-ISSN 1791-244X, Vol. 8, no 4, p. 427-431Article in journal (Refereed)
    Abstract [en]

    Until today, nineteen trinucleotide repeat expansions larger than forty repeat copies have been found in the human genome. Of these, the CAG/CTG repeat is predominant motif with twelve loci identified, ten of which have been associated with the development of neurodegenerative diseases. We have developed a cloning approach which isolates disease genes containing trinucleotide repeat expansions. The method is based on size separation of genomic fragments, followed by subcloning and library hybridization with an oligonucleotide probe. Fractions and clones containing expanded repeats are identified by the repeat expansion detection (RED) method throughout the cloning procedure. Large family materials are not required and as little as 10 microg genomic DNA from a single individual is sufficient for this method. Using this strategy we have cloned two DNA fragments containing expanded repeats from two unrelated patients with a clinical diagnosis of cerebellar ataxia. Sequencing of the two fragments showed sequence identities with two disease genes, the Huntington gene and the ataxin 3 gene, respectively. The method should be adaptable to the cloning of any long repeat motif in any species. Furthermore the experimental steps can be performed in less than a month, making it very effective and time efficient to disease gene identification.

  • 50.
    Zander, Cecilia Soussi
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Soussi, Thierry
    Breast-cancer stromal cells with TP53 mutations2008In: New England Journal of Medicine, ISSN 0028-4793, E-ISSN 1533-4406, Vol. 358, no 15, p. 1635-1635Article in journal (Other academic)
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