Denaturing gradient gel electrophoresis for the molecular characterization of six patients with guanidinoacetate methyltransferase deficiency

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. Three index patients with mutations confirmed by techniques other than DGGE [P1, P2, and P6; see Refs. (5), (8)] and three new patients (P3, P4, and P5) with undetectable GAMT activity but unknown GAMT deficiency mutations (9)(10) were investigated. Details of the patient studies are summarized in Table 1⇓ , and the locations and frequencies of the mutations identified in these patients are shown in Fig. 1⇓ . We extracted DNA from dried-blood spot, filter-paper samples and from fibroblasts and lymphoblasts using …