| 1. |
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| 2. |
- Heiskanen, M, et al.
(författare)
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A novel method to quantitate methylation of specific genomic regions
- 1994
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Ingår i: PCR methods and applications. - 1054-9803. ; 4:1, s. 26-30
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Tidskriftsartikel (refereegranskat)abstract
- A new solid-phase primer extension method has been developed for the quantitation of methylation differences and is described here. The method is less cumbersome than Southern blot analysis, expresses the results in a numerical format, can be adapted to a microtitration well format, and thus allows the analysis of a large series of samples. The model gene analyzed here is the calcitonin gene, but the method can be adapted to the analysis of methylation alterations in any area of the genome. The primer extension method clearly differentiated hypermethylated samples from normally methylated samples and a range for normal values could be determined. In quantitation experiments the method showed linearity in a range from 2% to 100% malignant blasts diluted with normal leukocytes.
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| 3. |
- Hietala, M, et al.
(författare)
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Prospects of carrier screening of aspartylglucosaminuria in Finland
- 1993
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Ingår i: European Journal of Human Genetics. - 1018-4813 .- 1476-5438. ; 1:4, s. 296-300
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Tidskriftsartikel (refereegranskat)abstract
- The frequency of carriers of the AGUFin mutation, the predominant mutation causing aspartylglucosaminuria in Finland, was determined in a population sample comprising 553 newborns from a delivery hospital in southern Finland, and 607 from a hospital in northern Finland. The AGUFin point mutation was identified from cord blood samples using the PCR-based, solid-phase minisequencing method. Nineteen carriers of the AGUFin mutation were detected, 8 (1:69) in the sample from the southern and 11 (1:55) from the northern population, respectively. The solid-phase minisequencing method proved to be rapid and convenient for the detection of the AGUFin mutation, and can readily be applied in large-scale carrier screening at the population level.
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| 4. |
- Ihalainen, J, et al.
(författare)
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Towards automatic detection of point mutations : use of scintillating microplates in solid-phase minisequencing
- 1994
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Ingår i: BioTechniques. - 0736-6205 .- 1940-9818. ; 16:5, s. 938-943
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Tidskriftsartikel (refereegranskat)abstract
- Simplification of molecular genetic techniques is one of the main features of large-scale clinical applications of mutation analysis. The solid-phase minisequencing method, which is based on single-nucleotide primer extension by a DNA polymerase on a solid support, is an easy way of detecting point mutations of previously known locations. Here the procedure was further simplified by the use of microplates made of scintillating plastics, a microplate format scintillation counter and an automatic microplate washer. DNA samples from patients with either a hereditary aspartylglucosaminidase (AGA) gene point mutation or an acquired N-ras gene mutation were analyzed by three different minisequencing detection procedures utilizing tritiated nucleotides. The new counting method with scintillating plates was compared to traditional liquid scintillation counting in scintillation vials or to another microplate format procedure, which requires addition of scintillation liquid. In all three methods, normal individuals, heterozygous carriers of the AGA mutation and homozygous patients could be unequivocally discriminated. The N-ras mutation in leukemic blasts could also be detected with high resolution. The coefficients of variation and reproducibility of the scintillating microplate method were almost identical to those of the traditional liquid scintillation assay, which was used as a reference method. The technical innovations adopted here for performing minisequencing assays reduce significantly the labor required without affecting the quality of the results.
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| 5. |
- Ikonen, E, et al.
(författare)
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Aspartylglucosaminuria : cDNA encoding human aspartylglucosaminidase and the missense mutation causing the disease
- 1991
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Ingår i: EMBO Journal. - 0261-4189 .- 1460-2075. ; 10:1, s. 51-58
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Tidskriftsartikel (refereegranskat)abstract
- We have isolated a 2.1 kb cDNA which encodes human aspartylglucosaminidase (AGA, E.C. 3.5.1.26). The activity of this lysosomal enzyme is deficient in aspartylglucosaminuria (AGU), a recessively inherited lysosomal accumulation disease resulting in severe mental retardation. The polypeptide chain deduced from the AGA cDNA consists of 346 amino acids, has two potential N-glycosylation sites and 11 cysteine residues. Transient expression of this cDNA in COS-1 cells resulted in increased expression of immunoprecipitable AGA protein. Direct sequencing of amplified AGA cDNA from an AGU patient revealed a G----C transition resulting in the substitution of cysteine 163 with serine. This mutation was subsequently found in all the 20 analyzed Finnish AGU patients, in the heterozygous form in all 53 carriers and in none of 67 control individuals, suggesting that it represents the major AGU causing mutation enriched in this isolated population. Since the mutation produces a change in the predicted flexibility of the AGA polypeptide chain and removes an intramolecular S-S bridge, it most probably explains the deficient enzyme activity found in cells and tissues of AGU patients.
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| 6. |
- Ikonen, E, et al.
(författare)
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Dissection of the molecular pathology of aspartylglucosaminuria provides the basis for DNA diagnostics and future therapeutic interventions
- 1993
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Ingår i: Scandinavian journal of clinical and laboratory investigation. Supplementum. - 0085-591X. ; 213, s. 19-27
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Tidskriftsartikel (refereegranskat)abstract
- Aspartylglucosaminuria (AGU) is exceptional among lysosomal storage diseases since it represents the only known amidase deficiency in man, being caused by an inadequate function of aspartylglucosaminidase (AGA, E.C. 3.5.1.26.). This amidase is essential in one of the final steps in the ordered breakdown of glycoproteins since it cleaves Asn from the residual N-acetylglucosamines (for reviews see 1, 2). The deficiency of the enzyme activity results in the typical lysosomal accumulation of the abnormal degradation products (mainly aspartylglucosamine, 2-acetamido-1-beta-L-aspartamido-1,2-dideoxyglucose) in patients' cells and tissues. The diagnosis of AGU has so far been based on the detection of abnormal metabolites in urine and decreased enzyme activity in the cultured fibroblasts or isolated lymphocytes. Prenatal diagnosis has been possible by demonstrating the deficient enzyme activity of amniocytes or chorion villus biopsies. Identification of carriers has been difficult and unreliable due to the high individual variation in AGA activity and prerequisite for isolated blood lymphocytes. During the past few years we have purified the human enzyme into homogeneity, isolated the full length cDNA and characterized the majority of AGU mutations in this cDNA. This work facilitated the development of a reliable DNA diagnostic test suitable also for large scale carrier screening. The molecular pathology of the most common AGU mutation was unravelled, this being a prerequisite for the oncoming developments for therapy. Although AGU is a relatively rare disease, characterization of the AGU mutations and their cellular consequences have revealed highly interesting new phenomena in the biosynthesis of this lysosomal enzyme, some of which carry general biological significance.
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| 7. |
- Ikonen, E, et al.
(författare)
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Quantitative determination of rare mRNA species by PCR and solid-phase minisequencing
- 1992
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Ingår i: PCR methods and applications. - : Cold Spring Harbor Laboratory. - 1054-9803. ; 1:4, s. 234-240
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Tidskriftsartikel (refereegranskat)abstract
- We present a new method for quantification of mRNA, in which the limitations of the current quantitative PCR methods can be overcome. A known amount of a synthetic RNA standard differing from the mRNA to be quantified by a single nucleotide is reverse-transcribed and amplified together with the mRNA template using a biotinylated primer. The biotinylated PCR product is immobilized on a streptavidin-coated solid support and denatured. The ratio between the two amplified sequences is determined by separate "mini-sequencing" reactions, in which a detection step primer annealing immediately adjacent to the site of the variable nucleotide is elongated by a single labeled dNTP complementary to the nucleotide at the variable site. The ratio between the incorporated labels accurately determines the ratio between the two sequences in the original RNA sample. We applied this method to quantify the mRNA of human aspartylglucosaminidase (AGA) in tissues and cultured cells. AGA is a lysosomal enzyme participating in the degradation of glycoproteins. A mutation in the AGA gene abolishes the enzyme activity and leads to aspartylglucosaminuria (AGU), a recessively inherited metabolic disorder. The mRNA quantification revealed that the normal and mutant genes are expressed at similar levels in kidney, liver, and cultured fibroblast, whereas the amount of AGA mRNA in normal placenta and brain is significantly higher than that found in the corresponding samples from AGU patients. The method presented here is generally applicable for PCR-based quantification of rare mRNAs and DNA as well.
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| 8. |
- Ikonen, E, et al.
(författare)
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Spectrum of mutations in aspartylglucosaminuria
- 1991
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - 0027-8424 .- 1091-6490. ; 88:24, s. 11222-11226
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Tidskriftsartikel (refereegranskat)abstract
- Aspartylglucosaminuria (AGU) is an inherited lysosomal storage disorder caused by the deficiency of aspartylglucosaminidase. We have earlier reported a single missense mutation (Cys163----Ser) to be responsible for 98% of the AGU alleles in the isolated Finnish population, which contains about 90% of the reported AGU patients. Here we describe the spectrum of 10 AGU mutations found in unrelated patients of non-Finnish origin. Since 11 out of 12 AGU patients were homozygotes, consanguinity has to be a common denominator in most AGU families. The mutations were distributed over the entire coding region of the aspartylglucosaminidase cDNA, except in the carboxyl-terminal 17-kDa subunit in which they were clustered within a 46-amino acid region. Based on the character of the mutations, most of them are prone to affect the folding and stability and not to directly affect the active site of the aspartylglucosaminidase enzyme.
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| 9. |
- Jalanko, A, et al.
(författare)
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Screening for defined cystic fibrosis mutations by solid-phase minisequencing
- 1992
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Ingår i: Clinical Chemistry. - 0009-9147 .- 1530-8561. ; 38:1, s. 39-43
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Tidskriftsartikel (refereegranskat)abstract
- We have developed a rapid method for the quantitative detection of point mutations and deletions. In this minisequencing method, enzymatically amplified DNA, 5'-biotinylated in one strand, is bound to a solid phase and denatured. A detection primer, constructed to end immediately before the mutation, is annealed to the immobilized single-stranded template and elongated with a single, labeled deoxynucleoside residue. We have applied the solid-phase minisequencing method to the detection of the major mutation, delta F508, causing cystic fibrosis (CF). In the presence of the allele with the delta F508 mutation, [3H]dTTP is incorporated; with the nonmutated allele, [3H]dCTP is incorporated. Thus, samples from heterozygous individuals allow the incorporation of both labels. The method was evaluated by analyzing 59 coded DNA specimens collected from 20 Finnish CF patients and their parents. The ratio of [3H]C to [3H]T gave unambiguously the allele combination. The solid-phase minisequencing method was also applicable to the analysis of three CF mutations simultaneously, i.e., delta F508, G542X, and G551D. We conclude that the microtiter-plate-based minisequencing test is an accurate method for the screening of defined sequence alterations in the CF gene.
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| 10. |
- Juvonen, V, et al.
(författare)
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Quantification of point mutations associated with Leber hereditary optic neuroretinopathy by solid-phase minisequencing
- 1994
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Ingår i: Human Genetics. - 0340-6717 .- 1432-1203. ; 93:1, s. 16-20
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Tidskriftsartikel (refereegranskat)abstract
- About two-thirds of patients with Leber hereditary optic neuroretinopathy (LHON) harbor mutations in mitochondrial DNA at positions 11778 (ND4) or 3460 (ND1). Thus, the clinical diagnosis of LHON can often be confirmed with mutation analysis. Detection of pathogenic mutations and quantification of heteroplasmy has mainly relied on PCR and restriction site analysis and densitometric scanning. We applied the recently developed solid-phase minisequencing method, based on primer-guided nucleotide incorporation, to the simultaneous detection and quantitation of the ND4/11778 and ND1/3460 mutations. The method was highly sensitive, heteroplasmy as low as 1.5% being easily detected. Rapid, reproducible, and accurate results prove solid-phase minisequencing to be the method of choice for quantitative analysis of LHON mutations.
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