| 1. |
- Elmgren, A, et al.
(author)
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DNA sequencing and screening for point mutations in the human Lewis (FUT3) gene enables molecular genotyping of the human Lewis blood group system.
- 1996
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In: Vox sanguinis. - 0042-9007. ; 70:2, s. 97-103
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Journal article (peer-reviewed)abstract
- The human Lewis gene encodes an alpha(1,3/1,4)-fucosyltransferase responsible for synthesis of the Le(a) and a Le(b) antigens. To define the molecular background for non-functional Lewis genes we have sequenced PCR-amplified DNA fragments from two Le(a-b-) individuals. One was homozygously mutated at nucleotides 202(T --> C) and 314 (C --> T), altering Trp68 to Arg and Thr105 to Met, and the other was homozygously mutated at nucleotides 59 (T --> G) and 1067 (T --> A), altering Leu20 to Arg and Ile356 to Lys. Using PCR we screened for these and additionally one other mutation at nucleotide 508 (G --> A) among 40 Caucasians. Of 15 Le(a-b-) individuals, 7 typed as le59/1067le202/314, 4 as le202/314le202/314 and 1 as le59/1067le59/1067. Of 21 Le(a-b+) and 4 Le(a+b-), 17 typed as LeLe and 7 as Lele202/314. A pedigree study of 8 Lewis-positive individuals showed that the mutations at nucleotides 202 and 314 were located on the same allele.
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| 2. |
- Fernandez-Mateos, P, et al.
(author)
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Point mutations and deletion responsible for the Bombay H null and the Reunion H weak blood groups.
- 1998
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In: Vox sanguinis. - 0042-9007. ; 75:1, s. 37-46
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Journal article (peer-reviewed)abstract
- OBJECTIVE: Definition of the molecular basis of the Reunion and the Bombay red cell and salivary H-deficient phenotypes. METHODS: Sequence and expression of FUT1 and FUT2 genes from H-deficient individuals. Family segregation analysis of the mutations responsible for the fucosyltransferase defects of H, secretor and Lewis systems. RESULTS: The Indian red cell H null Bombay phenotype depends on a new mutation of the FUT1 gene. T725-->G changing Leu242-->Arg. Their salivary nonsecretor phenotype is secondary to a complete deletion of the FUT2 gene. The red cell H weak Reunion phenotype depends on another new mutation of FUT1, C349-->T which induces a change of His117-->Tyr. Their salivary nonsecretor phenotype is due to the known Caucasian inactivating mutation G428-->A. CONCLUSION: Single prevalent FUT1 and FUT2 point mutations and a deletion are responsible for the Indian Bombay H null and the Reunion H weak phenotypes found on Reunion island. This is in contrast with other H-deficient phenotypes where sporadic nonprevalent inactivating mutations are the rule.
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| 3. |
- Larson, Göran, 1953, et al.
(author)
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Typing for the human lewis blood group system by quantitative fluorescence-activated flow cytometry: large differences in antigen presentation on erythrocytes between A(1), A(2), B, O phenotypes.
- 1999
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In: Vox sanguinis. - 0042-9007. ; 77:4, s. 227-36
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Journal article (peer-reviewed)abstract
- BACKGROUND: Lewis phenotyping by hemagglutination is an unreliable routine method for Lewis antigen designation. Now genomic typing of the Lewis gene is available. Additionally, flow cytometry has been used for typing. We wanted to compare the results of Lewis typing in healthy individuals using the three methods. MATERIALS AND METHODS: Ninety-three randomly selected plasma donors were genotyped for inactivating Secretor (FUT2) G428A and Lewis (FUT3) T59G, T202C, C314T, G508A and T1067A point mutations. All Le(a+b-) individuals (nonsecretors) were homozygous for the FUT2 G428A mutation and all Le(a-b-) individuals had inactivating mutations on both FUT3 alleles. Fixed erythrocytes were analyzed by fluorescence-activated flow cytometry and the results were compared with hem- agglutination and genotypic data. Antigen availability was expressed as median fluorescence intensity and as percentage positive cells with fluorescence intensities > or =10(2). RESULTS: Using an anti-Le(a) reagent a mean of 99% of erythrocytes from Le(a+b-) individuals and 1% of erythrocytes from Le(a-b-) or Le(a-b+) individuals were stained positive. Using an anti-Le(b) reagent, a mean of 71% of erythrocytes from A(1), 95% from B and 99% from O and A(2) Le(a-b+) individuals and less than 10% of erythrocytes from Le(a-b-) or Le(a+b-) individuals were stained positive. After papain treatment 100% of the erythrocytes from A(1) and A(1)B Le(a-b+) individuals stained positive without increase in background staining. The flow cytometric technique revealed large differences in staining intensities, within each ABO Le(a-b+) subgroup which was not directly correlated to plasma donation frequencies nor to Secretor or Lewis genotypes. CONCLUSION: Flow cytometry may prove valuable as a Lewis blood group typing technique but also as a research tool when investigating Lewis phenotypes of human erythrocytes.
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| 4. |
- Rydberg, Lennart, 1944, et al.
(author)
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An ELISA technique for quantitation of human xenoantibodies binding to pig cells: application in patients with pig kidneys extracorporeally connected to the circulation.
- 1998
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In: Xenotransplantation. - 0908-665X. ; 5:2, s. 105-10
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Journal article (peer-reviewed)abstract
- A quantitative ELISA technique for determination of human anti-pig xenoantibody number in serum samples has been established using pig lymphocytes and pig/rabbit erythrocytes as target cells and a pool of serum from human blood group AB donors. The number of low affinity antibodies binding to the cells was determined by quantitation following the use of aqueous washing of the cells and separation of bound and unbound antibodies with the phthalate oil method. The efficiency of different soluble Gal(alpha)1-3Gal-terminating di- and tri-saccharides to inhibit antibody binding was tested and found to vary between 70-90% at a saccharide concentration of 10 mg/ml. The assay was used to evaluate the antibody changes in two patients who, after plasmapheresis treatments, had pig kidneys extracorporeally connected to their blood circulation. The number of anti-pig IgM/IgG antibodies bound to each pig lymphocyte were reduced from 5,600/13,200 to 1,300/3,100 in patient 1 and from 1,200/6,500 to 500/2,100 in patient 2 by three consecutive daily plasmapheresis treatments. Although the lymphocytotoxic titers were reduced to very low levels, the antibody numbers still present in the blood of patient 1 caused a hyperacute rejection of the pig kidney. However, the antibody levels in patient 2 did not cause rejection of this kidney during 15 min perfusion time. A strong anti-pig antibody response 3 weeks after the perfusion experiment was found in patient 1 as shown by 27,600/245,300 IgM/IgG molecules bound to pig lymphocytes corresponding to an increase of lymphocytotoxic titer from 8 to 512. The second patient showed a much weaker immune response with 1,400/19,800 IgM/IgG antibodies corresponding to a lymphocytotoxic titer increase from 8 to 32. The use of this quantitation technique enables more accurate investigation of antibody binding to xenogenic target cells than conventional titration techniques.
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