Abstracts

RATIONALE: Our goals are to identify genes that increase risk for common human seizure disorders. Mouse QTL mapping studies from our labs identified distal chromosome one (chr 1) as harboring a gene of fundamental importance in mediating the difference in susceptibility between the relatively seizure resistant C57BL/6J (B6) and sensitive DBA/2J (D2) inbred mouse strains. We studied candidate genes from this genomic region to identify DNA variations associated with seizure sensitivity in mice and humans.
METHODS: A congenic strain approach has narrowed the critical interval on chr 1 to 5-10cM. Candidate genes mapped to this interval in mice (and human 1q21-23, 1q41-44) were studied by direct sequence comparison of coding regions. Total brain RNA was used to isolate cDNA clones via RT/PCR for sequencing. Mouse genomic clones were isolated by hybridization screening of a B6 BAC library (RPCI-23). Human gene sequences were amplified from genomic DNA extracted from blood samples collected from epilepsy patients and controls. DNA sequencing and electrophoretic assays including SSCP and RFLP were used to identify gene variations.
RESULTS: We identified a single nucleotide polymorphism (SNP C785G, 785bp from translation start) in the mouse KCNJ10 gene that alters amino acid 262 (of 379) from a Thr to a Ser. The Thr residue found in the B6 strain is conserved across species including human, rat, and rabbit. We surveyed inbred mouse strains and examined the relationship between KCNJ10 genotype and electrical seizure threshold (EST). Results show that B6 derived lines contain the Thr residue and have higher EST, whereas all other strains tested had the Ser residue and lower EST compared to B6. BAC clone 157J4 is being sequenced (GenBank [pound] AC074311) and results reveal the B6 KCNJ10 coding region to be intronless. However, portions of the 5[scquote] UTR are displaced some 30KB upstream from the start of translation. We have also identified variations in the human KCNJ10 gene. One SNP (C1037T, R271C, acc [pound] nm002241) was found to be associated with common human seizure disorders in a case control study design ([chi]2 = 5.76 p[lt] 0.023, n= 305 epilepsy/260 control individuals).
CONCLUSIONS: The biological relevance of the KCNJ10 T262S variation in mice awaits confirmation in transgenic animals to determine if the amino acid change affects EST. Likewise, the human association study awaits replication in an independent population. However, these data support the notion that mouse QTL mapping studies can identify genes involved in disease susceptibility and that the human homologs of those genes are excellent candidates for genetic association testing.
Support: NIH NS33243 and NS40554 to TNF & Center for Neurobiology and Behavior, University of PA.