MUTATION AND POLYMORPHIC MARKER ANALYSES OF 65K-GLUTAMATE AND 67K-GLUTAMATE DECARBOXYLASE GENES IN 2 FAMILIES WITH PYRIDOXINE-DEPENDENT EPILEPSY

Pyridoxine-dependent epilepsy is a disease inherited as an autosomal r ecessive trait, characterized by rapid response to pharmacological dos ages of pyridoxine. The defect has been suggested to reside in glutama te decarboxylase (GAD), since a mutant GAD with an abnormally high K-m for a cofactor, pyridoxal phosphate, could not synthesize an adequate amount of gamma-amino butyric acid [Scriver and Whelan (1969) Ann NY Acad Sci 166: 83]. To test this hypothesis, we studied two affected fa milies by screening for mutations in the GAD mRNA and by analyzing a p olymorphic marker in the GAD gene. Since two forms of GAD, GAD65 and G AD67, have been identified in human brain, we analyzed both forms. To overcome the limited accessibility of brain tissues, we utilized the m inute amounts of GAD mRNAs ectopically transcribed in lymphoblasts. Th e ectopic GAD transcripts were amplified by reverse-transcription-medi ated, nested polymerase chain reaction for mutation analysis. Two and three base substitutions were found in GAD65 and GAD67 cDNAs, respecti vely. All of them were, however, polymorphisms that were also found in control subjects. We then examined a (CA) repeat polymorphism in the GAD65 gene and found that different maternal alleles were transmitted to two affected sibs in one family. Thus, an etiological mechanism oth er than a K-m mutant GAD is responsible for pyridoxine-dependent epile psy.