Denaturing Gradient Gel Electrophoresis for the Molecular Characterization of Six Patients with Guanidinoacetate Methyltransferase Deficiency

Abstract
Guanidinoacetate methyltransferase [(GAMT); EC 2.1.1.2] deficiency is the first recognized inborn error of creatine biosynthesis, manifesting in infancy with severe neurologic symptoms such as epilepsy, mental retardation, muscular hypotonia, and progressive extrapyramidal movement disorder ( 1). Patients with GAMT deficiency have shown favorable responses to oral supplementation of creatine-monohydrate, but complete reversal of symptoms has not been observed ( 2). Biochemical findings include high urinary excretion of guanidinoacetate (the immediate precursor of creatine and substrate of GAMT), low urinary excretion of creatinine [conversion product of intracellular creatine; see Ref. ( 3)], and depletion of creatine in brain and muscle ( 4). After assessing two index patients ( 5)( 6), we aimed to establish methods for the noninvasive molecular diagnosis of GAMT deficiency. We recently developed a radiochemical method for the determination of GAMT activity in cultured skin fibroblasts and in virus-transformed lymphoblasts ( 7). Here we report a denaturing gradient gel electrophoresis (DGGE) technique for the screening of mutations in the GAMT gene in DNA extracted from dried-blood spot filter-paper samples and from fibroblasts and virus-transformed lymphoblasts.