A novel point mutation in the 3' region of the prothrombin gene at position 20221 in a Lebanese/Syrian family.
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3' Untranslated Regions↗
Biomedical subjects
K R Klingler
Publications and source records attributed to K R Klingler.
Apolipoprotein E polymorphism: automated determination of apolipoprotein E2, E3, and E4 isoforms.
Apolipoprotein E (apo E) plays an essential role in lipoprotein metabolism, where it is involved in the clearance of chylomicrons and very low density lipoproteins. Apart from some rare variants, apo E exists in three common isoforms (E2, E3, and E4). The different isoforms have not only been associated with different plasma lipid levels but have also been correlated with certain pathological conditions, such as lipid disorders (dysbetalipoproteinemia, hypercholesterolemia), cardiovascular diseases, and Alzheimer's disease. Here we describe a rapid, automated test for the determination of the most frequent polymorphisms (E2, E3, and E4). This polymerase chain reaction-based test allows the reliable discrimination of all six genotypes. The assay has been developed especially for the nonspecialized routine clinical laboratory by employing an analyzer and chemistry often present in this type of laboratory. Because of its low costs and easy handling, the assay can be performed on a daily basis.
Apolipoprotein E2↗
Haemochromatosis: automated detection of the two point mutations in the HFE gene: Cys282Tyr and His63Asp.
Hereditary haemochromatosis (HH) is one of the most common inherited diseases among Caucasians. Two mutations in the HFE gene have been implicated in HH: 80 to 90% of the patients with HH are homozygous for the point mutation CYS282Tyr, while the majority of the remaining patients displays either a compound heterozygosity for the mutation CYS282Tyr and the point mutation HIS63Asp, or are homozygous for HIS63Asp. Though the disease can be treated easily, symptoms are non-specific, and onset and severity are influenced by environmental factors, and therefore the disease can remain undetected until decades of iron overload lead to irreversible damage in a variety of organs, which may result in their failure. In order to detect patients with HH, simple and cost-effective tests are needed. We have developed a rapid, automated, PCR-based test which makes use of a diagnostic restriction site in each of two amplified fragments. The test employs off-the-shelf chemistry and uses the automated detection process of an immunoassay analyzer that is available in many clinical laboratories, thus avoiding an additional investment in a more specialized PCR analyzer. Because of its low costs and easy handling, the assay is particularly suited for the routine clinical laboratories.
Aspartic Acid↗
Apolipoprotein B-100: employing the electrochemiluminescence technology of the Elecsys systems for the detection of the point mutation Arg(3500)Gln.
Apolipoprotein B-100 (apo B-100) plays an essential role in lipoprotein metabolism where it is involved in the clearance of LDL particles from the bloodstream. The mutation Arg3500Gln in the apo B-100 gene impairs the binding of the LDL particles to the LDL receptor, resulting in elevated LDL-cholesterol levels in the blood which, in turn, fuel the development of premature atherosclerosis. Here we describe a rapid, automated test for the detection of the most frequent mutation in the apo B-100 gene. This PCR-based test employs electrochemiluminescence as detection technology and allows the reliable discrimination of all genotypes. The assay has been especially developed for the non-specialized routine clinical chemistry laboratory by employing an analyzer and chemistry often present in this type of labof1tory. Because of its low costs and easy handling the assay can be performed on a daily basis.
Amino Acid Substitution↗
Activated protein C (APC)-resistance: automated detection of the point mutation at position 1691 in the factor V gene.
Proteolytic cleavage of factor Va, caused by activated protein C, is an important mechanism in limiting clot formation in normal haemostasis. A single point mutation in the factor V gene has been demonstrated to cause resistance of factor Va to proteolytic cleavage by activated protein C. With an 8-fold increased risk of thrombosis and a 2 to 13% prevalence in the Caucasian population for the heterozygous state of this mutation, knowledge of the patient's genetic disposition is of great importance in conditions such as pregnancy, surgery, use of oral contraceptives and immobilization. Therefore we have developed an automated test for the detection of the factor V mutation. This PCR based test makes use of the disappearance of an Mnl 1 restriction site if the mutation is present. The assay has been developed for the widely used ES-systems of Boehringer Mannheim. The test discriminates between the heterozygous and the homozygous state. Because of its low costs and easy handling the assay can be used as a screening test and can be performed in routine clinical laboratories.
Activated Protein C Resistance↗
Activated protein C resistance: automated detection of the factor V Leiden mutation by mismatch hybridization.
BACKGROUND: A single point mutation in the factor V gene has been demonstrated to be the cause of factor Va resistance to proteolytic cleavage by activated protein C. Knowledge of the patient's genetic disposition is of great importance in situations such as pregnancy, surgery, use of oral contraceptives, and immobilization. METHODS: We have developed a rapid, automated test for the detection of the factor V mutation that makes use of differences in thermal stability between perfect-match and non-perfect-match hybrids. A DNA fragment spanning the mutation is amplified with a biotin-labeled primer. Ruthenium-labeled oligonucleotides, perfectly matching either the biotinylated wild-type strand or the biotinylated mutation strand, are added. Heating to 95 degrees C and subsequent cooling lead to the formation of double-stranded DNA. Under the conditions chosen, ruthenium-labeled oligonucleotides form stable, double-stranded DNA with the biotinylated strand only if both strands perfectly match each other. The ruthenium signal is measured on a modified Elecsys 1010 system (Roche Diagnostics). RESULTS: The ratio between the signals obtained with perfectly matching and non-perfectly matching oligonucleotides reflects the genetic status. Analyzed samples can be divided into three nonoverlapping groups based on these ratios. We confirmed the reliability of the method by analyzing several samples of known genetic status; the results were identical in every single instance. CONCLUSIONS: The test discriminates unambiguously between the heterozygous and the homozygous states. Because of its low costs and easy handling, the assay is suitable for use in routine laboratories of clinical chemistry.
Autoanalysis↗