In 1995 we mapped the ADPEAF locus to a 10-cM region on chromosome 10q24 in a single extended pedigree2
. Linkage was subsequently reported to an overlapping interval in another large family, narrowing the minimal genetic region to approximately 3 cM, assuming the causative gene was the same4
. Analysis of additional pedigrees confirmed the linkage but failed to narrow the region further5–7
. To screen for disease-related mutations, we resequenced all coding-exon and bordering-intron sequences from positional candidate genes in the overlap interval in one affected individual from each of three ADPEAF pedigrees showing linkage to chromosome 10q24 (families 6610, A and B; )2,7
. We then genotyped putative disease-related mutations in all available family members from the three linked pedigrees, all family members from two smaller families with ADPEAF (families C and D; ) and 123 unrelated control individuals.
Fig. 2 Segregation of putative disease alleles in families with ADPEAF. Family 6610 is the original family used to establish linkage between 10q24 DNA markers and disease2. Filled bars mark the presence and boundaries of disease-related haplotypes, defined in (more ...)
Resequencing of LGI1
identified presumptive mutations in each of the five families with ADPEAF ( and ). All tested affected individuals from the five families harbored a single copy of a putative disease mutation, as did all obligate carriers and individuals classified as ‘unknown’ who were found to carry the disease-linked haplotype (). Several unaffected individuals also carried the disease haplotype and presumptive mutation. Whether these individuals manifest subclinical signs of disease or have undergone recent changes in affection status is not yet known, but the results are consistent with our previous estimate of 71% disease-gene penetrance in family 6610 (ref. 2
LGI1 mutations in families with ADPEAF
To distinguish disease-related mutations from polymorphic variants, we analyzed a panel of unrelated control individuals by resequencing the entire coding region of exon 8, all of exon 6 and all of exons 3 and 4 plus intronic sequences, each in a total of 123 control individuals. Resequencing revealed several polymorphisms (none encoding amino-acid changes), but none of the five putative mutations was detected.
Three mutations predictably altered the codon frame, leading to missense mutations and premature truncation of the Lgi1 protein, whereas one point mutation in exon 8 (family D) changed a glutamic acid residue to an alanine (). This amino-acid residue, as well as those extending ten residues upstream and three residues downstream, was conserved in the only identifiable, full-length LGI1 homolog, the highly conserved mouse Lgi1 (data not shown). One putative mutation occurred in intronic DNA, changing a single nucleotide at the third base from the acceptor intron–exon junction of exon 4 (; family B). We speculated that the alteration would either lead to aberrant splicing or, if left unspliced, encode a stop codon at amino-acid residue 118, leading to truncation of the protein (). We carried out RT–PCR on mRNA isolated from lymphoblasts prepared from a control individual and four affected individuals from family B. PCR amplification spanning the region between exons 3 and 6 identified one normal and one aberrant band in affected individuals but not in the control sample (). DNA sequencing of this aberrant band showed that the IVS3(–3)C→A alteration led to retention of the entire intron 3 in a portion of the LGI1 transcript produced in individuals who carried this putative mutation ().
Fig. 3 Predicted effect of ADPEAF mutations on Lgi1. a, Structural organization of LGI1. LGI1 spans 36.9 kb and consists of eight exons ranging in size from 72 bp (exons 2–5) to 1,197 bp (exon 8). The mRNA transcript consists of a 262-bp 3′ UTR (more ...)
Fig. 4 Aberrant LGI1 splicing in family B. a, LGI1 oligonucleotide primers specific for exon 3 and exon 6 were amplified using RT–PCR and mRNA isolated from lymphoblastoid cell lines. Lane 1, control sample; lanes 2–5, samples from four affected (more ...)
Chernova et al
were the first to describe LGI1
, upon observing that the gene was disrupted by translocation in the T98G glioblastoma multiforme (GBM) cell line and in over one-quarter of primary tumors. LGI1
expression is absent or significantly downregulated in many high-grade but not low-grade gliomas, suggesting a role for LGI1
in glial tumor progression8,9
. The functional inactivation of LGI1
in high-grade GBM tumors provides the only evidence of the protein’s function. Assuming that LGI1
functions primarily as a tumor progressor as opposed to a tumor suppressor, mutations in LGI1
would predictably increase the severity or progression of glial tumors rather than increase glial tumor frequency. Although we found no clear cases of glioblastomas in these families with ADPEAF, one affected male died of a brain tumor 18 months after his diagnosis. GBM is the most common malignant tumor of the adult central nervous system and has a median post-treatment survival of less than two years. Unfortunately, the affected individual died over 30 years ago and all records and samples have been discarded.
The genomic structure of LGI1
and the highly conserved mouse homolog (97% amino-acid homology) were recently reported9
; a modified representation of the human gene is depicted in . The predicted protein structure consists of a 5′ signal peptide and three functional leucine-rich repeats (LRRs), flanked by cysteine-rich repeat sequence clusters. The carboxy terminus shows no significant homology to any known protein other than the mouse homolog and is composed of two direct tandem repeats and a possible transmembrane domain. All LRR-containing proteins seem to be involved in ligand binding or protein–protein interactions, and at least half are involved in signal transduction pathways10,11
. The presence of four cysteine residues in each of the LRR flanking repeat sequences may indicate that LGI1
belongs to the largest group in the LRR superfamily, the adhesive proteins and receptors10
. It seems that LGI1
encodes a protein consisting of an extracellular domain with LRR repeat motifs, a transmembrane segment and an intracellular segment of unknown function. The extracellular portion of Lgi1 aligns most closely with a small group of proteins including the Drosophila
proteins slit, toll and tartan, and the mammalian Trk
gene family, in which the LRRs are known to bind nerve growth factor and other neurotrophins10–12
The genes tartan
seem to be essential in the development of the central nervous system. The tartan protein presumably functions as a membrane-bound cell-adhesion protein, and slit as a diffusible inhibitory cue for both neuronal growth-cone guidance and neuronal migration. The Drosophila
slit protein is produced in midline glial cells where the LRR motif is required for the directional guidance of neuronal migration13,14
. The Drosophila
and vertebrate slit
genes are involved both in mechanisms of growth-cone guidance and migration of entire neurons15
. Although there is no direct evidence of functional conservation among LGI1
and these LRR-containing genes, the role of slit
, and perhaps tartan
, in neuronal cell migration and neuronal growth-cone guidance is consistent with a presumptive role for LGI1
in both epilepsy and tumor metastasis.
In humans, LGI1
is expressed primarily in brain (cerebellum, cortex, medulla, occipital pole, frontal lobe, temporal lobe and putamen) and muscle and at lower levels in spinal cord (data not shown)8
. Mouse Lgi1
was also predominantly expressed in fetal brain but not in fetal lung, liver or kidney, as detected by northern-blot analysis of whole mouse embryos on embryonic days 7, 11, 15 and 17 (data not shown).
To determine whether mouse Lgi1 mRNA was predominantly expressed in neurons or glial cells, we carried out high-resolution, chromogenic RNA in situ analysis (). The expression pattern of Lgi1 was predominantly neuronal and was consistent with our current understanding of the anatomic regions involved in temporal lobe epilepsy. Mouse Lgi1 was expressed in a highly specific pattern in the neocortex and limbic regions. In the hippocampus, the amount of expression was highest in the granule cells of the dentate gyrus, the large-bodied cells within the hilus of the dentate gyrus and the pyramidal cells of the CA3 region, and much lower in the CA1 region. Lgi1 was also expressed in the amygdala, the piriform cortex and in distinct laminae of the dorsal lateral cortex, including the auditory cortex of mice. The degree of expression of Lgi1 varied among different brain nuclei and among individual cells within distinct brain nuclei (), indicating a finely tuned transcriptional regulation and raising the possibility that differences in the amount of Lgi1 produced by individual cells are of functional importance.
Fig. 5 Analysis of Lgi1 expression in the adult mouse brain by chromogenic RNA in situ hybridization. a–c,f, An antisense riboprobe (a–c) or a sense (control) riboprobe (f) of Lgi1 was hybridized to 16-μm thin coronal cryosections of (more ...)
All of the genes previously identified as causing idiopathic epilepsy syndromes in humans have been voltage-gated or ligand-gated ion channels16
, and only one gene with a different (but unknown) mechanism has been reported in a mouse model17
is not homologous to any known ion channel gene. Although there are no direct experimental data addressing the function of LGI1
, its homology to other genes encoding LRR-containing proteins and its presumptive role in glioma tumor progression suggest a role in cell–cell communication. It is intriguing to speculate whether LGI1 functions like slit and tartan,
or other LRR-containing adhesion proteins, in neuronal-cell migration and axon growth-cone guidance in the developing central nervous system, such that loss of a single gene copy would interfere with normal neuronal development and lead to focal seizures and other symptoms of ADPEAF. Loss of both LGI1
gene copies might lead to glial cell metastasis by upsetting neuron–glia homeostasis, perhaps through loss of neuronal inhibition of glial cell proliferation or differentiation.
We have reported five putative disease mutations in five families with ADPEAF2,3,7
. Based on segregation patterns in affected families, predicted effects of mutations on protein sequence and lack of detection in control samples, it is likely that several, if not all, of the candidate mutations lead to temporal lobe epilepsy through haploinsufficiency. Although preliminary data indicate that loss of both copies of human LGI1
promotes glial tumor progression, it is not clear from the current study how the homozygous loss of a predominantly neuronal gene produces this effect. Such an effect is clearly possible, however, because neurons are known to inhibit glial mitosis, and interactions between neurons and glia apparently establish precisely regulated homeostasis in both tissues.