Abstracts

Because GABRB3 deficient mice shows absence like features including EEG characteristics and pharmacological response because atypical absences in Angelman syndrome correlates with deletions which includes GABRB3, and because Transmission Disequilibrium Test showed possible genetic association between GABRB3 and CAE, we screened for mutations in GABRB3 in families with CAE. We detected two heterozygous missense mutations (P11S, S15F) in the alternative signal peptide, exon1a of GABRB3 segregating with CAE affected members of three Hispanic families. The same missense mutations were not present in 34 other probands with CAE and 440 Hispanic healthy controls Here, we report on the functional consequences of these missense mutations., We used mammalian expression vectors, pCMV-SPORT 6 into which was cloned exon1-9 of GABRB3 and pFP-N1 [GFP fusion vector]. We amplified the mutation containing Exon1a with designed primers with restriction enzyme sites, and replaced Exon1 in pCMV-SPORT6 with Exon1a with/without mutations. The construct which has the region from exon1a to exon9 without stop codon was amplified from the replaced vector. After digestion, each construct was cloned into pFP-N1. HeLa cells were transfected with expression constructs and examined 48 hours post-transfection and homogenized. Protein samples were run on a 10% Tris-HCl gel and transferred onto a nitrocellulose filter. After blocking, the filter was processed through sequential incubations with the primary antibody for 1 h, and then with horseradish secondary antibody for 1h. Immunoreactive proteins on the filter were visualized using Typhoon software 9410., Immunoblot studies revealed that both P11S and S15F mutations in exon1a decreased the expression of the protein level when compared with controls. The average densitometric image quantitation was 33%([plusmn]0.014) of control for P11S and 69%([plusmn]0.14) of control for S15F, normalized to actin density(N=3)., Our results indicate that both P11S and S15F mutations in exon1a decrease expression of protein levels and suggest less trafficking of the nascent peptide from the endoplasmic reticulum to cell membrane. GABRB3 is a main component of GABAR in reticular nucleus of thalamus and expressed transiently in other thalamic nuclei in the perinatal period, decreasing in adult. We speculate that the decreased expression of GABRB3 in developmental brain leads to absence seizures.,