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 (Praw = 0.000035 and Pgenome-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.
Table 1.
Four highly-associated SNPs spread across the linked 1.7 Mb homozygosity region, together with the c.1552A>T mutation, were genotyped and tested for association from 28 BFJE cases and 112 healthy controls.
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.