Diagnosis of N-acetylglutamate synthase deficiency by use of cultured fibroblasts and avoidance of nonsense-mediated mRNA decay.
Molecular diagnosis of N-acetylglutamate synthase deficiency (NAGSD) has become possible now that the corresponding gene has been identified. We describe the genetic analysis of a patient with NAGSD using low-level transcripts derived from cultured fibroblasts. One defective allele (c.1306-1307insT) was detected by PCR amplification. However, the transcript from a second mutation (IVS3 - 2A>T), causing aberrant splicing with the generation of a premature termination codon, was not detected until interference of nonsense-mediated mRNA decay was abrogated by the translation inhibitor cycloheximide. We demonstrate that low-level transcripts in cells that do not express significant enzyme activity are a valuable tool for molecular studies of genetic alterations, and suggest routine abrogation of nonsense-mediated mRNA decay using cycloheximide when transcript analysis is performed.
Häberle
J
J
Universitätsklinikum Münster, Klinik und Poliklinik für Kinderheilkunde, Münster, Germany. haeb@uni-muenster.de
Denecke
J
J
Schmidt
E
E
Koch
H G
HG
D000123:Acetyltransferases / Q000172:deficiency*; D000483:Alleles; D000592:Amino Acid Metabolism, Inborn Errors / Q000175:diagnosis* / Q000201:enzymology / Q000235:genetics*; D051046:Amino-Acid N-Acetyltransferase; D002478:Cells, Cultured; D003062:Codon / Q000235:genetics; D018389:Codon, Nonsense / Q000235:genetics*; D003513:Cycloheximide; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D005347:Fibroblasts / Q000201:enzymology*; D006801:Humans; D007231:Infant, Newborn; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics
D003062:Codon; D018389:Codon, Nonsense; D018076:DNA, Complementary; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D003513:Cycloheximide; D000123:Acetyltransferases; D051046:Amino-Acid N-Acetyltransferase; C494874:NAGS protein, human