Appendix e-1: Methods
Patient assessment. Eleven family members were personally interviewed by specifically asking about déjà-vu, auditory or other auras. The Partial Seizure Symptom Definitions were used as set of definitions (e1). All of patients were blood sampled for genetic analysis and underwent neurological examination and prolonged video-EEG recordings during wakefulness and sleep. Neuropsychological evaluation was undertaken in patients who agreed to participate and comprised evaluation of estimated intelligence (Progressive Matrices, Series 1), language lateralization (Hand Edinburgh laterality quotient, Language Laterality Index, Repetition task), and assessment of verbal production (fluency and naming tests) and comprehension (discrimination, lexicon, complex commands tasks at the Italian Battery for assessing Aphasic Deficits (BADA; e2)). Patients’ performance was compared with Italian norms.
Molecular genetic studies.DNA was extracted from blood by standard methods and LGI1 exons were PCR amplified as described in Ref. 2.Sequencing of PCR products was performed using the Big Dye Terminator Cycle Sequencing kit (ABI PRISM, Applied Biosystems) and an ABI3730 automatic sequencer (Applied Biosystems). Prediction of pathogenic effect of the mutation was assessed by the PolyPhen programme ()
Cell transfection assay. Cell transfection assay was performed as described (e3). Briefly, LGI1 wild type or LGI1 1219C>T expression constructs containing a Flag peptide in frame with the LGI1 cDNA sequence were transfected into human embryonic kidney 293 (HEK293) cells. Twenty-four hours after transfection cells were washed and then overlaid with the serum-free medium Opti-MEM. After about 20 hours, cells were lysed and the medium was collected and concentrated about 20x using Centricon YM30 concentrators (Millipore). Aliquots of cell lysates and concentrated media were loaded on an SDS-PAGE gel and analysed by western blot using the anti-Lgi1 antibody ab30868 (Abcam) or the anti-Flag antibody F7425 (Sigma-Aldrich).
In silico modelling of Lgi1 EPTP domain. The human Lgi1 sequence (accession code: O95970) and aligned homologous sequences were retrieved from OMA database (e4). The 3D model of the EPTP domain of Lgi1 was built using the structure of WD repeat protein (WDR5; PDB code: 2GNQ) structure (e5) as described in Leonardi et al. (in preparation). The model was built following the method used in Sommer et al. (e6). Given the problematic nature of repeated sequences, the initial model was used as a starting point for manual refinement using knowledge about the approximate location of repeats, key residues and secondary structure
e-References
e1. Choi H, Winawer MR, Kalachikov S, et al. Classification of partial seizure symptoms in genetic studies of the epilepsies. Neurology2006;66:1648-1653.
e2. Miceli G, Laudanna, A, Burani C, et al. Analisi dei Deficit Afasici-BADA, Cepsag. Rome: Università Cattolica del Sacro Cuore, 1994.
e3. Furlan S, Roncaroli F, Forner F, et al. The LGI1/Epitempin gene encodes two protein isoforms differentially expressed in human brain. J Neurochem 2006;98:985-991.
e4. Schneider A, Dessimoz C, et al. OMA Browser - Exploring Orthologous Relations across 352 Complete Genomes. Bioinformatics 2007;23:2180-2182.
e5. Schuetz A, Allali-Hassani A, Martín F, et al. Structural basis for molecular recognition and presentation of histone H3 by WDR5. EMBO J 2006;25:4245-4252.
e6. Sommer I, Toppo S, Sander O, et al. Improving the quality of protein structure models by selecting from alignment alternatives. BMC Bioinformatics. 2006;27:364.