LGI2 truncation causes a remitting focal epilepsy in dogs

Abstract

One quadrillion synapses are laid in the first two years of postnatal construction of the human brain, which are then pruned until age 10 to 500 trillion synapses composing the final network. Genetic epilepsies are the most common neurological diseases with onset during pruning, affecting 0.5% of 2-10-year-old children, and these epilepsies are often characterized by spontaneous remission. We previously described a remitting epilepsy in the Lagotto romagnolo canine breed. Here, we identify the gene defect and affected neurochemical pathway. We reconstructed a large Lagotto pedigree of around 34 affected animals. Using genome-wide association in 11 discordant sib-pairs from this pedigree, we mapped the disease locus to a 1.7 Mb region of homozygosity in chromosome 3 where we identified a protein-truncating mutation in the Lgi2 gene, a homologue of the human epilepsy gene LGI1. We show that LGI2, like LGI1, is neuronally secreted and acts on metalloproteinase-lacking members of the ADAM family of neuronal receptors, which function in synapse remodeling, and that LGI2 truncation, like LGI1 truncations, prevents secretion and ADAM interaction. The resulting epilepsy onsets at around seven weeks (equivalent to human two years), and remits by four months (human eight years), versus onset after age eight in the majority of human patients with LGI1 mutations. Finally, we show that Lgi2 is expressed highly in the immediate post-natal period until halfway through pruning, unlike Lgi1, which is expressed in the latter part of pruning and beyond. LGI2 acts at least in part through the same ADAM receptors as LGI1, but earlier, ensuring electrical stability (absence of epilepsy) during pruning years, preceding this same function performed by LGI1 in later years. LGI2 should be considered a candidate gene for common remitting childhood epilepsies, and LGI2-to-LGI1 transition for mechanisms of childhood epilepsy remission.

Figure 1. Mapping and identification of the benign focal juvenile epilepsy mutation in Lagotto Romagnolo.

A) Genome-wide association analysis maps the disease locus to CFA3 with the strongest association at a SNP at position 89,159,216 (P_raw  = 0.000035 and P_genome-wide  = 0.08). B) A 1.7 Mb homozygous block spanning from 87.3 Mb to 89.0 Mb is present in affected dogs (bottom set of 11 dogs) and not in unaffected dogs (top set of 11 dogs). C) Sequencing of the Lgi2 coding regions revealed a homozygous c.1552A>T mutation that causes a premature stop codon in exon 8, resulting in truncation of the last 12 amino acids in the affected cases.

Figure 2. Expression of the mutant Lgi2 transcript is normal.

Total RNA extracted from blood of a healthy (c.1552A/A), a carrier (c.1552A/T), and an affected (c.1552T/T) Lagotto Romagnolo, as well as from the cerebellum of a healthy (1552A/A) Saluki (referred as SB) was transcribed into cDNAs for amplification by PCR with Lgi2 exon-specific primers. The truncation mutation does not alter expression level of Lgi2.

Figure 3. Mutant LGI2 is not secreted in cell cultures.

HEK293 cells were transfected with human V5-tagged wt and p.K534X mutant LGI2 clones. Aliquots of lysed cells (lanes 1&2) and culture media (lanes 3&4) were harvested, concentrated, and analyzed with anti-V5 and anti-GADPH antibodies to follow expression of the recombinants. Only wild-type LGI2 protein is found in culture media indicating that the K534X mutation prevents secretion.

Figure 4. Wild-type (wt) LGI2, like LGI1, binds to ADAM22 and ADAM23 on the cell surface.

Indicated cDNAs were co-transfected into COS7 cells, and after 24 hours surface-bound LGI1-Flag and LGI2-V5 (red) were labeled before cell permeabilization and staining of the HA-tagged ADAM22 (A) and ADAM23 (B) proteins (green). Wt Lgi1 and LGI2 bound to both cell surface ADAM receptors, whereas mutant LGI2 was not secreted and did not bind the receptors.

Figure 5. LGI2 interacts with ADAM22 and ADAM23 in rat brain.

Western blotting shows that antibodies against ADAM22 or ADAM23 co-precipitate Lgi2 in brain lysates.

Figure 6. Developmental expression of LGI2 in mouse brain.

Mouse cerebelli (A) and forebrains (B) were harvested postnatally every other day for the first four weeks of life (except days 5 and 17) and Lgi2 transcript levels were measured by quantitative RT-PCR. Lgi2 expression is at the highest levels in the forebrain at birth but decreases significantly after p13. In the cerebellum Lgi2 levels remain stable. Trendline is shown by dashed lines.