Principle investigator

Prof. Dr. med. Ortrud Steinlein


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Identification and functional analysis of genes in human idiopathic epilepsy

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Introduction
Idiopathic epilepsies are those epilepsies in which a symptomatic background is neither detected nor suspected, but a genetic etiology is likely or proven. Most idiopathic epilepsies are caused by ion channel mutations, implicating an etiology based on imbalances in synaptic transmission or neuronal excitability 1. However, since 2002, three genes have beeen identified which do not code for ion channels, but their mutations also cause idiopathic epilepsies. Among them, LGI1 was the first identified gene in this group, its mutations cause a rare epilepsy syndrome called autosomal dominant lateral temporal lobe epilepsy (ADLTE) 2. Mutations in Mass1 were identified in a mouse model of audiogenic epilepsy3. Last year, EFHC1 was found to be associated with human juvenile myoclonic epilepsy (JME), a subtype of idiopathic generalised epilepsies(IGE)4.
The etiology of  the epilepsies caused by these three non-channel genes is still unclear. Our group has been concentrating on studying the function of the two human genes LGI1 and EFHC1 using biochemical methods and zebrafish model, as well as trying to identify further non-channel epilepsy genes by looking for candidate genes with similar structure and same evolutionary origin.

Fig 1
Fig 1: Unrooted phylogeny of the LGI sub-families. Branch lengths are drawn in proportion to the expected number of nucleotide substitutions per codon.

Results
Phylogeny of the vertebrate LGI gene family
Our earlier work showed that LGI1 belongs to a subfamily of leucine-rich repeat genes comprised of four members (LGI1-LGI4) in mammals5. In this study, both comparative developmental as well as molecular evolutionary methods were applied to investigate the evolution of the LGI gene family and, subsequently, of the functional importance of its different gene members.
We identified LGI genes with BLAST searches and RT-PCR experiments in chimpanzee, mouse, rat, chick, zebrafish and puffer fish genomes. No LGI ortholog was found in  the invertebrate genomes of nematodes, the fruitfly and the ascidian, Ciona intestinalis. Our data suggest that the LGI gene family originated in the evolutionary lineage leading to the vertebrates.The phylogeny of the LGI gene family (Fig1) indicates an origin of the gene family through two rounds of gene or genome duplications. In this scenario, each of the two gene pairs LGI1/LGI4 and LGI2/LGI3 had one ancestral precursor gene. These two ancestral genes themselves may have arisen from a common "proto-LGI" gene. LGI4 appears to be absent from zebrafish and puffer fish. The most probable scenario is that LGI4 was lost in the lineage leading to the ray-finned fish. Alternatively, LGI4 may have originated from a duplication of LGI1 in the lineage leading to the sarcopterygians (lobe-finned fish), and also the mammals.

Fig 2
Fig 2: Expression of lgi1 paralogs. 

Expression pattern of the zebrafish lgi genes
We examined the embryonic expression patterns of all zebrafish lgi genes by whole-mount in situ hybridisation. Expression of lgi1a is first evident in the ventral diencephalon and at 24 hours post fertilisation (hpf) strong expression is observed in the developing eyes, in the ventral midbrain and hindbrain, and in the peripheral spinal cord (Fig.2A-D). By 48 hpf lgi1a is strongly expressed in the retinal ganglion cell layer, the diencephalon and along the ventral aspect of the
hindbrain (Fig. 2E-H). Notably, all lgi1a expression domains are in neural tissues. lgi1b is expressed at 24 hpf in presumptive telencephalic and diencephalic bands and cranial paraxial mesenchyme. At 48 hpf, lgi1b transcripts are detected in the optic tectum, the cerebellum and in the zone of migrating neurons that proliferated in the rhombic lip. Expression is further observed in the dorsal thalamus and in the retinal ganglion cell layers (Fig. 2J-L). Overall, lgi1a expression is predominant in ventral parts of the mid- and hindbrain, while lgi1b is more dorsally restricted in this region. In situ-PCRs on adult transversal brain sections (Fig.2M,N) show that lgi1a and lgi1b are expressed in the outer layer of the periventricular grey zone of the optic tectum, an area rich in tectal neurons. lgi1b, in addition, is strongly expressed in the cerebellum. Both genes colocalize with nuclear areas of ganglion cells. At this level of resolution we could not detect expression in adult brain glial cells.

Fig 3
Fig 3: Expression of lgi2 and lgi3 genes.

In contrast, expression of both lgi2 paralogs is generally restricted to a few cells of putative (neuro-) ectodermal origin during embryogenesis. Both genes are expressed in trigeminal ganglion cells and in a few cells in the posterior head (Fig. 3A-C). More prominently, lgi2a is transiently detectable in dorsal spinal cord neurons. Finally, lgi3 is expressed in cranial mesodermal cells and in a few cells on each side of the otic vesicle (Fig. 3D and not shown). lgi3 appears to be co-expressed with lgi1a in the peripheral spinal cord in one- and two-day old embryos, and is detected in a reiterated symmetrical pattern of cells in the ventral hindbrain (Fig. 3E-F).

Evolution of the vertebrate LGI gene family
Vertebrate LGI genes from mammals and teleosts were tested for signs of natural positive or negative selection in coding regions. Interestingly, LGI1 and LGI4 orthologs show evidence for strong negative natural selection (purifying selection), while the remaining groups of LGI orthologs exhibited rather moderate signs of negative selection pressure. Purifying selection is the form of natural selection that acts to eliminate selectively deleterious replacement mutations. Our data support the assumption that purifying selection is indicative of a gene that is more likely to cause human disease and points out the possible significance of LGI4 for human neurological diseases. Interestingly, six out of ten amino acids mutated in human ADLTE exhibit high selection pressures, an observation which is in agreement with the role these mutations are assumed to play in the pathogenesis of this rare epilepsy.

Identification of EFHC2, the paralog of EFHC1
We identified and cloned a human paralog of EFHC1, which we named EFHC2. EFHC2 is localised on chromosome Xp11.3  and is the only EFHC1 paralog in human genome. Their protein products share an overall similarity of 41,6% at the amino acid level. The lacking of  conservation of exon-intron boundaries between EFHC1 and EFHC2 argues that the assumed duplication event creating the two EFHC genes already happened before the vertebrate lineage. It’s shown that massive intron gain and loss might accompany major evolutionary transitions, whereas little if any intron gains and limited amount of intron loss occur in the vertebrates. The ancient origin of both genes is also supported by the fact that homologs (i.e. Rib72) of EFHC1/EFHC2 are found in species evolutionary as far away as Chlamydomonas.

Analysis  of EFHC2 as a candidate suscepbility gene for  IGE
To test whether genetic variation of the EFHC2 gene region confers susceptibility to JME and other common IGE syndromes, we carried out an association study of six single nucleotide polymorphisms of the EFHC2 gene in 654 German IGE patients and 662 population controls. Because of its X-chromosomal localisation, male and female patients were analysed seperately. None of the genotype distributions in female controls deviated significantly from that expected by Hardy-Weinberg equilibrium (P > 0.15). The scoring rate was >99% for the study samples. Genotyping reliability was ensured on duplicated samples (6.5%) and consistent Mendelian inheritance was demonstrated in eight control families. The LD pattern between adjacent SNPs in male controls showed week LD between SNP rs5906926 in the 5´-region of the EFHC2 gene and SNP rs1885293 in intron 1 (D´= 0.08), whereas strong LD between the five intragenic SNPs (D´ > 0.95) indicates that these SNPs are located within a single haplotype block. Haplotype analysis of the five intragenic SNPs revealed four common haplotypes (> 3%) in the controls, accounting for > 93% of all haplotypes in male controls.
In males, the frequency of the T allele (Y430) of the amino acid substitution polymorphism (S430Y; rs2208592) was significantly increased in the JME patients (14.4%) compared to the controls (7.2%; #2 = 4.705, df = 1, P = 0.030; OR = 2.17; 95-CI: 1.06 – 4.43). This allelic association was even stronger for 81 males with "classical" JME (JME without absence seizures) (#2 = 6.06, df = 1, P = 0.014; OR = 2.46; 95-CI: 1.18 – 5.13). No allelic association of the Y430 allele was observed for the entire group of IGE males (#2 = 0.305, df = 1, P = 0.58; OR = 1.19; 95-CI: 0.64 – 2.20). None of the allele frequencies of the other SNPs differed significantly between the IGE groups when compared with the controls (P > 0.18). Likewise, no significant difference in the distribution of the common intragenic five-SNP haplotypes was found in the IGE groups compared with the controls (P > 0.18). The observed allelic association between JME and Y430 was no longer significant after Bonferroni adjustment for multiple comparisons.
In females, there was no significant difference in the allele frequencies of any SNP between the IGE groups when compared with the controls (P > 0.21). Likewise, a similar distribution of the common intragenic five-SNP haplotypes was found in the IGE groups as in the controls (P > 0.82).
An association with the gonosomal gene EFHC2 would be in accordance with the observed preponderance of maternal inheritance in JME. Independent replication studies are needed to further analyse the tentative association described here.

Outlook
The expression analysis of LGI genes in zebrafish provides the basis for the planned morpholino knock-down experiments, with which the question of LGI function can be adressed directly in a simplified although still considerably conserved vertebrate nervous system. Besides the evolutionary and expression pattern analysis of the LGI gene family, we are also studying the function of LGI1 protein with biochemical methods. Using yeast two-hybrid screenings, two candidate proteins interacting with LGI1 protein have been identified. We are currently verifying them with immuno-precipitation assays. Also in this sense, zebrafish offers an attactive model system to test the biochemical results in vivo. The EFHC genes are being studied in zebrafish model as well. As a summary, by combining molecular genetical, biochemical and zebrafish model mehtods, we hope to get further insight into the function of the non-channel epilepsy genes and to  understand the etiology of the epilepsies caused by their mutations.

Lit.: 1. Steinlein OK. Genetic mechanisms that underlie epilepsy. Nat. Rev. Neurosci. 5:400-8. 2. Kalachikov S et al. Mutations in LGI1 cause autosomal-dominant partial epilepsy with auditory features.Nat Genet. 2002 30: 335-41. 3. Skradski SL et al. A novel gene causing a mendelian audiogenic mouse epilepsy.Neuron. 2001 31:537-44. 4. Suzuki T et al. Mutations in EFHC1 cause juvenile myoclonic epilepsy.Nat Genet. 2004 36:842-9. 5. Gu W et al. The LGI1 gene involved in lateral temporal lobe epilepsy belongs to a new subfamily of leucine-rich repeat proteins. FEBS Lett. 2002 519:71-6.