Abstract
Ferraro TN, Golden GT, Smith GG, Martin JF, Lohoff FW, Gieringer TA, Zamboni D, Schwebel CL, Press DM, Kratzer SO, Zhao H, Berrettini WH, Buono RJ
Mamm Genome 2004;15(4):239–251
Previous quantitative trait loci (QTL) mapping studies document that the distal region of mouse chromosome (Chr) 1 contains a gene(s) that is in large part responsible for the difference in seizure susceptibility between C57BL/6 (B6) (relatively seizureresistant) and DBA/2 (D2) (relatively seizure-sensitive) mice. We now confirm this seizure-related QTL (Szs1) using reciprocal, interval-specific congenic strains and map it to a 6.6-Mb segment between Pbx1 and D1Mit150. Haplotype conservation between strains within this segment suggests that Szs1 may be localized more precisely to a 4.1-Mb critical interval between Fcgr3 and D1Mit150. We compared the coding region sequences of candidate genes between B6 and D2 mice using RT-PCR, amplification from genomic DNA, and database searching and discovered 12 brain-expressed genes with SNPs that predict a protein amino acid variation. Of these, the most compelling seizure-susceptibility candidate is Kcnj10. A survey of the Kcnj10 SNP among other inbred mouse strains revealed a significant effect on seizure sensitivity such that most strains possessing a haplotype containing the B6 variant of Kcnj10 have higher seizure thresholds than those strains possessing the D2 variant. The unique role of inward-rectifying potassium ion channels in membrane physiology coupled with previous strong association between ion channel gene mutations and seizure phenotypes puts even greater focus on Kcnj10 in the present model. In summary, we confirmed a seizure-related QTL of large effect on mouse Chr 1 and mapped it to a finely delimited region. The critical interval contains several candidate genes, one of which, Kcnj10, exhibits a potentially important polymorphism with regard to fundamental aspects of seizure susceptibility.
Buono RJ, Lohoff FW, Sander T, Sperling MR, O'Connor MJ, Dlugos DJ, Ryan SG, Golden GT, Zhao H, Scattergood TM, Berrettini WH, Ferraro TN
Epilepsy Res 2004;58(2–3):175–183
PURPOSE: Our research program uses genetic linkage and association analysis to identify human seizure sensitivity and resistance alleles. Quantitative trait loci mapping in mice led to identification of genetic variation in the potassium ion channel gene Kcnj10, implicating it as a putative seizure-susceptibility gene. The purpose of this work was to translate these animal model data to a human genetic association study.
METHODS: We used single-stranded conformation polymorphism (SSCP) electrophoresis, DNA sequencing, and database searching (NCBI) to identify variation in the human KCNJ10 gene. Restriction fragment length polymorphism (RFLP) analysis, SSCP, and Pyrosequencing were used to genotype a single-nucleotide polymorphism (SNP, dbSNP rs#1130183) in KCNJ10 in epilepsy patients (n = 407) and unrelated controls (n = 284). The epilepsy group comprised patients with refractory mesial temporal lobe epilepsy (n = 153), childhood absence (n = 84), juvenile myoclonic (n = 111), and idiopathic generalized epilepsy not otherwise specified (IGE-NOS, n = 59), and all were of European ancestry.
RESULTS: SNP rs#1130183 (C> T) alters amino acid 271 (of 379) from an arginine to a cysteine (R271C). The C allele (Arg) is common, with conversion to the T allele (Cys) occurring twice as often in controls compared with epilepsy patients. Contingency analysis documented a statistically significant association between seizure resistance and allele frequency, Mantel–Haenszel χ2 = 5.65, df = 1; P = 0.017; odds ratio, 0.52; 95% CI, 0.33–0.82.
CONCLUSIONS: The T allele of SNP rs#1130183 is associated with seizure resistance when common forms of focal and generalized epilepsy are analyzed as a group. These data suggest that this missense variation in KCNJ10 (or a nearby variation) is related to general seizure susceptibility in humans.
Commentary
The researchers first found genetic variation in the Kcnj10 gene by using the mouse as a model for epilepsy. The epilepsy trait in mice was measured by determining the electrical current required to induce a seizure, that is, the maximal electroshock seizure threshold (MEST) test. MEST testing has been widely used as a model for epilepsy. C57BL6 mice are relatively resistant and therefore have high seizure thresholds, whereas DBA2 mice are seizure sensitive and have low seizure thresholds. These two inbred mouse strains with differing thresholds to electro-convulsive shock were crossed, and the Szs1 locus was mapped to chromosome 1 by using quantitative trait loci (QTL) analysis (1). The Szs1 locus accounts for a large proportion of the difference in seizure susceptibility between C57BL6 and DBA2 mice and also is associated with increased susceptibility to kainic acid–induced seizures (2) and pentylenetetrazol-induced seizures (3).
Specific genes responsible for QTL are notoriously difficult to pinpoint. In part, the problem is that most quantitative traits are due to many genes of small effect, and epilepsy is no different. Another difficulty is that the QTL region is often very large. In the present report, Ferraro et al. used congenic strains (C57BL6 background with a DBA2 chromosome 1 or vice versa) with overlapping portions of chromosome 1 to refine the Szs1 locus. MEST testing of these congenic strains refined the Szs1 locus to a 4.1-megabase interval, containing approximately 100 genes. Four genes in this region were particularly interesting to the investigators because they are directly involved in ion transport; the findings included two potassium channel genes, Kcnj9 and Kcnj10. With the full sequence of both C57BL6 and DBA2 available, it was a relatively simple matter to identify all the single-nucleotide polymorphisms (SNPs) in the region that varied between the two strains and also changed an amino acid in the protein. The list was further narrowed to 12 genes by selecting only those genes that are expressed in the brain, and Kcnj10 was still on the list. The possibility that one of the other 11 candidate genes identified is involved in seizure susceptibility cannot be ruled out.
The SNP identified in Kcnj10 was examined in 15 different inbred mouse strains, and the researchers discovered that only the C57-related strains had a threonine at residue 262 of Kcnj10; all non-C57 mice had a serine. When these strains were tested by using the MEST test, strains with the threonine generally had higher seizure thresholds, that is, were resistant to seizures. The threonine was present in all species where sequence was available, except in non–C57-related mice. Kcnj10 is a member of the inwardly rectifying potassium channel (Kir) family and is an attractive epilepsy candidate; however, the SNP detected may not be responsible but merely tightly linked to another causative gene. No physiologic studies were presented, and it would be interesting to test Kir current recordings from the different mouse strains to determine whether the threonine–serine substitution alters channel function.
The results provided convincing evidence for Kcnj10 being involved in altered seizure threshold in mice. In their second article, Buono et al. describe variation in KCNJ10 in patients with epilepsy. They found an arginine–cysteine variant at residue 271 of the protein, just nine amino acids away from the SNP described in mice. Cysteine is the less frequent allele and was present at a higher frequency in controls (7.9%) compared with epilepsy patients (4.2%), implying that the presence of the cysteine in the KCNJ10 protein confers resistance to epilepsy. The P value (P = 0.017) for this association was statistically significant but must be confirmed in a separate population. The authors concluded that variation in KCNJ10 is associated with multiple seizure types, rather than with any particular type of epilepsy. This seems to contrast with other reports, which demonstrated that genetic variation is associated with particular seizure disorders (4).
The results from the mouse and human studies provide strong evidence that the KCNJ10 gene can confer resistance to epilepsy. In almost every published epilepsy family in which a discrete gene has been identified, several members of the family inherit the defective gene but do not have epilepsy. It would be interesting to test these nonpenetrant family members for the cysteine allele of KCNJ10, because this may explain why some people can inherit an epilepsy gene without developing seizures.
Potassium channels are the most diverse group of the ion channel family and are important both in shaping the action potential and in neuronal excitability and plasticity. The two main classes of potassium channels are voltage gated (Kv) and inwardly rectifying (Kir) channels. Mutations in voltage-gated potassium channels have previously been associated with benign familial neonatal seizures (5). KCJN10 (the protein is also known as Kir4.1) is an inwardly rectifying channel that helps regulate extracellular potassium ion concentrations. KCNJ10 is expressed widely in the brain, predominantly by glial cells, with particularly high levels in the brainstem (6). Neurons become hyperexcitable when extracellular potassium levels are too high or too low (outside the 2-to 5-mM range). Potassium released from neurons is absorbed by astrocytes, through Kir channels, and redistributed. Homozygous Kcnj10 knockout mice die shortly after birth (7) and are probably not a good model for the more subtle genetic variation attributed to the seizure susceptibility. Both the mouse and human KCNJ10 variants are within a region involved with ionic conductance, channel subunit dimerization, and anchoring to the plasma membrane (8). Any or all of these functions may be subtly affected by the amino acid substitutions present in the mouse and human potassium channels. Functional studies will help determine if the variants cause alteration of channel conductance.
Growing evidence exists that Kir channels play an important role in epileptogenesis and may provide novel targets for the development of new antiepileptic drugs. Studies on the weaver mouse were the first to demonstrate that Kir channels are involved in seizure generation (9). This mouse line has a point mutation in the pore region of the Kcnj6 gene (also known as Girk2 orKir3.2). Studies in humans have so far failed to demonstrate association between KCNJ6 and epilepsy (10). Knockout of Kcnj11 (Kir6.2) also causes increased seizure susceptibility in mice (11). Polymorphisms in KCNJ3 have been associated with epilepsy in humans (10), and reduced Kircurrent has been reported in surgical tissue from patients with intractable temporal lobe epilepsy (11). The addition of KCNJ10 to the list of Kirchannels associated with seizures emphasizes the importance of this gene family in epileptogenesis.
The two reports by Buono, Ferraro, and colleagues are excellent examples of QTL analysis in mice translating into identification of an epilepsy susceptibility gene in humans. It is an exciting prospect that a single gene may be associated with resistance to multiple seizure types and may aid in the development of new treatments. However, caution must be maintained until these results can be confirmed. Confirmation will require functional demonstration that the sequence variants have a physiologic effect, as well as the testing of an independent epilepsy population for the presence of the cysteine variant. With the growing molecular resources available for both mice and humans, this type of success story will become more common. In the future, when sequencing individual genomes becomes commonplace, we may be able to determine a person's epilepsy risk based on the presence of certain genetic variants, such as those found in KCNJ10. An individual's likelihood of developing epilepsy will probably be determined by a combination of many different susceptibility and resistance genes, in addition to environmental factors.