| 1. |
- Grahn, Ammi, 1961, et al.
(författare)
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Determination of Lewis FUT3 gene mutations by PCR using sequence-specific primers enables efficient genotyping of clinical samples.
- 2001
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Ingår i: Human mutation. - : Hindawi Limited. - 1098-1004 .- 1059-7794. ; 18:4, s. 358-9
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Tidskriftsartikel (refereegranskat)abstract
- We have developed a polymerase chain reaction method using sequence-specific primers (PCR-SSP) for rapid and correct genotyping of the common Lewis (FUT3) gene mutations 59T>G, 202T>C, 314C>T, 508G>A, and 1067T>A. The PCR-SSP method was validated on 20 healthy blood donors and 16 non-insulin-dependent diabetic patients. All individuals were in parallel genotyped by our established polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The FUT3 genotypes, determined with the PCR-SSP method, were in complete accordance with the results of the PCR-RFLP reference method. The PCR-SSP method could also be adapted to assign the presence of a specific mutation to the respective FUT3 alleles. We found the method to be reliable, rapid and cheap with no requirements for restriction enzyme processing.
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| 2. |
- Andersson Shams Hakimi, Caroline, et al.
(författare)
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In vitro assessment of platelet concentrates with multiple electrode aggregometry.
- 2015
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Ingår i: Platelets. - : Informa UK Limited. - 0953-7104 .- 1369-1635. ; 26:2, s. 132-137
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Tidskriftsartikel (refereegranskat)abstract
- ABSTRACT Storage impairs platelet function. It was hypothesized that multiple electrode aggregometry in vitro could be used to follow aggregability in platelet concentrates over time and that the results predict the efficacy of platelet transfusion in an ex vivo transfusion model. In vitro platelet aggregability was assessed in apheresis and pooled buffy coat platelet concentrates (BCs) (n = 13 each) using multiple electrode aggregometry with different agonists 1, 3, 5 and 7 days after preparation. In the ex vivo transfusion model, whole blood samples from nine healthy volunteers were collected every second day. The samples were supplemented with stored platelets (+146 × 10(9) × l(-1)) from the same unit 1, 3, 5 and 7 days after preparation. Platelet aggregability was assessed in the concentrate and in the whole blood samples before and after platelet supplementation. There was a continuous reduction in in vitro platelet aggregability over time in both apheresis and pooled BCs. The same pattern was observed after ex vivo addition of apheresis and pooled BCs to whole blood samples. The best correlation between in vitro aggregability and changes in aggregation after addition was achieved with collagen as agonist (r = 0.67, p
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| 3. |
- Bucardo, Filemon, et al.
(författare)
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Genetic susceptibility to symptomatic norovirus infection in Nicaragua. : norovirus susceptibility in Nicaragua
- 2009
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Ingår i: Journal of medical virology. - : Wiley. - 1096-9071 .- 0146-6615. ; 81:4, s. 728-35
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Tidskriftsartikel (refereegranskat)abstract
- Host genetic resistance to Norovirus (NoV) has been observed in challenge and outbreak studies in populations from Europe, Asia, and USA. In this study, we have investigated if histo-blood group antigens can predict susceptibility to diarrhea caused by NoV in Nicaragua, Central America, and if this can be reflected in antibody-prevalence and titer to NoV among individuals with different histo-blood group antigen phenotypes. Investigation of 28 individuals infected with NoV and 131 population controls revealed 6% of non-secretors in the population and nil non-secretors among patients infected with NoV, suggesting that non-secretors may be protected against NoV disease in Nicaragua. Surprisingly, 25% of the population was Lewis negative (Le(a-b-)). NoV infections with genogroup I (GI) and GII occurred irrespective of Lewis genotype, but none of the Lewis a positive (Le(a + b-)) were infected. The globally dominating GII.4 virus infected individuals of all blood groups except AB (n = 5), while the GI viruses (n = 4) infected only blood type O individuals. Furthermore, O blood types were susceptible to infections with GI.4, GII.4, GII.7, GII.17, and GII.18-Nica viruses, suggesting that secretors with blood type O are susceptible (OR = 1.52) and non-secretors resistant. The overall antibody-prevalence to NoV GII.3 VLP was 62% with the highest prevalence among blood type B carriers (70%) followed by A (68%) and O (62%). All four investigated individuals carrying blood type AB were antibody-negative. Among secretors, 63% were antibody-positive compared to 33% among non-secretors (P = 0.151). This study extends previous knowledge about the histo-blood group antigens role in NoV disease in a population with different genetic background than North American and European.
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| 4. |
- Carlsson, Beatrice, et al.
(författare)
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The G428A nonsense mutation in FUT2 provides strong but not absolute protection against symptomatic GII.4 Norovirus infection. : Novel GII.4 disease pattern
- 2009
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Ingår i: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 4:5
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Tidskriftsartikel (refereegranskat)abstract
- In November 2004, 116 individuals in an elderly nursing home in El Grao de Castellón, Spain were symptomatically infected with genogroup II.4 (GII.4) norovirus. The global attack rate was 54.2%. Genotyping of 34 symptomatic individuals regarding the FUT2 gene revealed that one patient was, surprisingly, a non-secretor, hence indicating secretor-independent infection. Lewis genotyping revealed that Lewis-positive and negative individuals were susceptible to symptomatic norovirus infection indicating that Lewis status did not predict susceptibility. Saliva based ELISA assays were used to determine binding of the outbreak virus to saliva samples. Saliva from a secretor-negative individual bound the authentic outbreak GII.4 Valencia/2004/Es virus, but did not in contrast to secretor-positive saliva bind VLP of other strains including the GII.4 Dijon strain. Amino acid comparison of antigenic A and B sites located on the external loops of the P2 domain revealed distinct differences between the Valencia/2004/Es and Dijon strains. All three aa in each antigenic site as well as 10/11 recently identified evolutionary hot spots, were unique in the Valencia/2004/Es strain compared to the Dijon strain. To the best of our knowledge, this is the first example of symptomatic GII.4 norovirus infection of a Le(a+b-) individual homozygous for the G428A nonsense mutation in FUT2. Taken together, our study provides new insights into the host genetic susceptibility to norovirus infections and evolution of the globally dominating GII.4 viruses.
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| 5. |
- Gerin, I., et al.
(författare)
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ISPD produces CDP-ribitol used by FKTN and FKRP to transfer ribitol phosphate onto alpha-dystroglycan
- 2016
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Mutations in genes required for the glycosylation of alpha-dystroglycan lead to muscle and brain diseases known as dystroglycanopathies. However, the precise structure and biogenesis of the assembled glycan are not completely understood. Here we report that three enzymes mutated in dystroglycanopathies can collaborate to attach ribitol phosphate onto a-dystroglycan. Specifically, we demonstrate that isoprenoid synthase domain-containing protein (ISPD) synthesizes CDP-ribitol, present in muscle, and that both recombinant fukutin (FKTN) and fukutin-related protein (FKRP) can transfer a ribitol phosphate group from CDP-ribitol to alpha-dystroglycan. We also show that ISPD and FKTN are essential for the incorporation of ribitol into alpha-dystroglycan in HEK293 cells. Glycosylation of alpha-dystroglycan in fibroblasts from patients with hypomorphic ISPD mutations is reduced. We observe that in some cases glycosylation can be partially restored by addition of ribitol to the culture medium, suggesting that dietary supplementation with ribitol should be evaluated as a therapy for patients with ISPD mutations.
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| 6. |
- Grahn, Ammi, 1961, et al.
(författare)
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A novel mutation on the transferrin gene abolishes one N-glycosylation site and alters the pattern of transferrin isoforms, mimicking that observed after excessive alcohol consumption
- 2016
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Ingår i: Clinical Biochemistry. - 0009-9120. ; 49:6, s. 511-513
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVES: In the process of obtaining a driver's license, a healthy 28year old man presented increased levels of disialo-transferrin (TF) (approx. 20%, ref. value<2) by HPLC analysis of TF isoforms (%,CDT), while other markers of excessive alcohol consumption (PEth, MCV and gamma-GT) were in the normal range. The objective of this study was to determine the cause of the increased %,CDT levels. DESIGN AND METHODS:Serum TF isoforms were re-analyzed by LC-MS. All coding exons of the TF gene were Sanger sequenced. RESULTS:Analysis of TF isoforms by LC-MS confirmed the presence of increased disialo-TF and revealed a discrepancy in the mass difference between disialo-TF and tetrasialo-TF which suggested the presence of a genetic TF isoform with one abolished N-glycosylation site. Sanger sequencing of the TF gene revealed the presence of two missense mutations in heterozygous form: c.1295A>G (p.N432S) and c.1765C>T (p.P589S). p.N432S is a novel mutation that abolishes one N-glycosylation site of TF, while p.P589S is the polymorphism that defines the C2 isoform of TF. The sum of mass shifts caused by both amino acid substitutions agrees with the mass shift observed by LC-MS, which indicates that both variants are located in cis. CONCLUSIONS:An individual initially suspected of alcohol abuse based on elevated %CDT was shown to be carrier of a novel mutation in the TF gene that abolishes the N-glycosylation site at position p.N432. The presence of this genetic variant has to be kept in mind when interpreting TF isoform patterns.
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| 7. |
- Grahn, Ammi, 1961, et al.
(författare)
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Glycobiology and cancer
- 2009
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Ingår i: Encyclopedia of Cancer. - Berlin : Springer Verlag. - 9783540476481
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Bokkapitel (övrigt vetenskapligt/konstnärligt)
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| 8. |
- Grahn, Ammi, 1961
(författare)
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Human α2,3-sialyltransferases. Structure and Function of Alternatively Spliced Transcripts
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The recruitment of human peripheral blood leukocytes to sites of infection and inflammation requires the surface expression of Sialyl Lewis x glycoconjugates (SLex) on white blood cells and their interaction with E- and P-selectins on activated endothelial cells. Human ST3Gal III, ST3Gal IV and ST3Gal VI genes code for a2,3?sialyltransferases potentially involved in the biosynthesis of the SLex epitope. In order to characterize these a2,3-sialyltransferases we have cloned and sequenced RNA transcripts of these genes. Our clones have revealed an unexpected heterogeneity in transcript isoforms. Among our ST3Gal IV clones we isolated nine alternatively spliced transcripts covering the coding region of the human ST3Gal IV gene. Five of these isotranscripts have not been described before. Within our ST3Gal III clones we isolated 26 different transcripts from peripheral blood leukocytes and fetal brain, which showed a wide variety of deletions from 45 to 896 nucleotides, and insertions of 26 to 173 nucleotides. Among the insertions we identified two new exons (E3, E6). Eight of the ST3Gal III isotranscripts coded for potentially active enzymes. ST3GalVI en mening??In order to investigate if the alternatively spliced isotranscripts were specific for human peripheral blood leukocytes, we analyzed by RT-PCR and laser-induced fluorescent capillary electrophoresis (LIF-CE) the expression in twenty-one human tissues. We found a highly tissue specific expression of both ST3Gal III and ST3Gal IV isotranscripts. For the ST3Gal III gene, neural and muscular tissues showed the most complex patterns and were distinctly different from all other tissues examined.We stably transfected HEK 293 cells with six of the potentially enzymatically active isoforms of ST3Gal III. Structural differences in the isoenzymes were limited to exon losses in the stem regions of these enzymes. Crude cell extracts from all transfected clones showed a2,3-sialyltransferase activities preferentially towards the type 1-chain disaccharide precursor (Galb1,3GlcNAc) but with different efficiencies and with varying specificities for the type 2-chain (Galb1,4GlcNAc) and Galb1,3GalNAc precursors. Other splice variants of ST3Gal III, unlikely to express enzymatic activities, but clearly exhibiting a very tissue specific expression, may have other biological functions but this remains to be clarified.The RT-PCR-LIF-CE technique, as we have used it, has an unique capacity to identify alternatively spliced transcripts with minor sequence differences as well as sufficient reproducibility and sensitivity for qualitative and semi-quantitative analysis of gene transcripts not provided by other methods. We have shown that redundancy of critical sialyltransferase activities not only appears between different genes but also between different isoenzymes obtained through alternative splicing of the coding sequence of such genes.
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| 9. |
- Grahn, Ammi, 1961, et al.
(författare)
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Identification of seven new alpha2,3-sialyltransferase III, ST3Gal III, transcripts from human foetal brain
- 2004
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Ingår i: Glycoconj J. ; 20:7-8, s. 493-500
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Tidskriftsartikel (refereegranskat)abstract
- We have recently cloned and sequenced 19 human ST3Gal III gene isotranscripts from peripheral blood leukocytes and identified very complex patterns of isotranscripts of this gene in neuronal tissues. We have now cloned and sequenced additionally seven new isotranscripts from foetal brain. These novel isotranscripts showed losses of complete exons along the whole length of the coding sequence. None of the new isotranscripts coded for proteins with the two (L- and S-) sialylmotifs intact. One of the isotranscripts belonged to the isoform ST3Gal III -B, five to the ST3Gal III -C isoform and one to ST3Gal III -D isoform of isotranscripts, which lacks exon 3, exons 3 and 4 and exon 4 respectively. Two of the C series isotranscripts, ST3Gal III C4 and C11 had both lost exons 12 and 13 containing the S-motif but had otherwise the L- and the VS-motifs intact. Three isotranscripts, ST3Gal III C5, C12 and D5, were similar in the 3'-end coding for an identical amino acid sequence unrelated to the original enzyme. Isotranscripts ST3Gal III C9 and B10 were distinctly different from all other forms identified so far. The splice variants reported here are unlikely to express enzymatic activities but may other biological functions.
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| 10. |
- Hesse, Camilla, et al.
(författare)
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The N-terminal domain of α-dystroglycan is released as a 38kDa protein and is increased in cerebrospinal fluid in patients with Lyme neuroborreliosis.
- 2011
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Ingår i: Biochemical and biophysical research communications. - : Elsevier BV. - 1090-2104 .- 0006-291X. ; 412:3, s. 494-499
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Tidskriftsartikel (refereegranskat)abstract
- α-Dystroglycan is an extracellular adhesion protein that is known to interact with different ligands. The interaction is thought to stabilize the integrity of the plasma membrane. The N-terminal part of α-dystroglycan may be proteolytically processed to generate a small 38kDa protein (α-DG-N). The physiological significance of α-DG-N is unclear but has been suggested to be involved in nerve regeneration and myelination and to function as a potential biomarker for neurodegenerative and neuromuscular diseases. In this report we show that α-DG-N is released into different body fluids, such as lachrimal fluid, cerebrospinal fluid (CSF), urine and plasma. To investigate the significance of α-DG-N in CSF we examined the levels of α-DG-N and known neurodegenerative markers in CSF from patients diagnosed with Lyme neuroborreliosis (LNB) and healthy controls. In untreated acute phase LNB patients, 67% showed a significant increase of CSF α-DG-N compared to healthy controls. After treatment with antibiotics the CSF α-DG-N levels were normalized in the LNB patients.
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