pyrophosphate and Genetic-Diseases--Inborn

Loop-mediated isothermal amplification (LAMP) has several advantages: this technique involves gene amplification under isothermal conditions using only one high-specificity enzyme; the amplification efficiency is so high that large quantities of pyrophosphoric acid are formed as a by-product of DNA synthesis; furthermore, the results can be judged directly on the basis of turbidity. On the other hand, a PCR requires approximately 3.5-4.0 hours. The LAMP method is faster than the PCR method and is also relatively inexpensive. In the present study, we modified the composition of the reaction solutions to reduce the LAMP reaction time; more specifically, a thickener, either polyethylene glycol 8000 or 20000, was added. These results showed that the LAMP method was faster than the original method, and it is able to detect both turbidity and fluorescence. In conclusion, the LAMP reaction could be performed within 20 minutes when reaction mixture supplemented with a thickener was used. This method can be used for tests in various fields such as the diagnosis of hereditary disease and identification of viral infections as point-of-care testing.

Topics: Diphosphates; DNA; Genetic Diseases, Inborn; Humans; Nucleic Acid Amplification Techniques; Point-of-Care Systems; Polyethylene Glycols; Polymerase Chain Reaction; Solutions; Time Factors; Virus Diseases

Hypophosphatasia (HPP) is the instructive rickets or osteomalacia caused by loss-of-function mutation(s) within TNSALP, the gene that encodes the "tissue nonspecific" isoenzyme of alkaline phosphatase (TNSALP). HPP reveals a critical role for this enzyme in skeletal mineralization. Increased extracellular levels of pyridoxal 5'-phosphate and inorganic pyrophosphate (PP(i)) demonstrate that TNSALP is a phosphomonoester phosphohydrolase and a pyrophosphatase that hydrolyzes much lower concentrations of natural substrates than the artificial substrates of laboratory assays. Clearly, TNSALP acts at physiological pH and "alkaline phosphatase" is a misnomer. Aberrations of vitamin B(6) metabolism in HPP revealed that TNSALP is an ectoenzyme. PP(i) excesses cause chondrocalcinosis and sometimes arthropathy. The skeletal disease is due to PP(i) inhibition of hydroxyapatite crystal growth extracellularly so that crystals form within matrix vesicles but fail to enlarge after these structures rupture. Trials of alkaline phosphatase replacement therapy for HPP suggest that TNSALP functions at the level of skeletal tissues.

Topics: Alkaline Phosphatase; Animals; Diphosphates; Genetic Diseases, Inborn; Humans; Hypophosphatasia; Isoenzymes; Mice; Mice, Knockout; Phosphates; Pyridoxal Phosphate