Abstracts

Rationale: Protocadherin-19 (PCDH19)-clustering epilepsy (PCE) is a severe developmental and epileptic encephalopathy (DEE) and one of the most common monogenic epilepsies. It is characterized by cognitive impairment and intractable seizure clusters with onset in the first few years of life. PCDH19 is an X-linked gene that encodes a transmembrane cell adhesion molecule critical for cell-cell interactions during brain development. PCE affects females and rare mosaic males, while hemizygous males expressing only mutant PCDH19 do not develop epilepsy. A leading hypothesis to explain this phenomenon is that it occurs due to cellular interference associated with random X-inactivation (or mosaic mutations) in which cells expressing only wild type and those expressing only mutant PCDH19 fail to interact properly during brain development.

Methods: To test this hypothesis, we crossed PCDH19-null female mice with X-linked GFP reporter males. Female offspring (PCDH19^+/-/X-GFP) express GFP on the wild-type allele and no fluorescent marker on the PCDH19-null allele, allowing for easy visualization of wild-type and mutant cell populations. Wild-type C57/Bl6J mice were bred with X-linked GFP reporter males to produce control females (PCDH19^+/+/X-GFP). Prolonged EEG recordings were performed to evaluate for spontaneous seizures. In addition, as fever is a common trigger for seizure clusters in patients with PCE, we sought to evaluate seizure susceptibility to hyperthermia in our model. The body temperature of P15-16 female mice was gradually raised with a heat lamp and maintained at 42.5ºC for 15 minutes. Seizures were scored on a modified Racine scale by an observer blinded to genotype. PCE and control mouse brains were processed and evaluated for histological analysis of GFP expression patterns.

Results: Brains of PCE mice revealed a striking segregation pattern of PCDH19+ and null neurons (after random X-inactivation) in the cortex, hippocampal CA1 region and in interneuron progenitors in the ganglionic eminences (Fig. 1). Prolonged EEG recordings did not reveal spontaneous seizures in PCE or control mice. However, we found a lowered seizure threshold (Fig. 2a) and more severe seizures (Fig. 2b) following hyperthermia exposure in PCE mice compared to age matched controls. Preliminary evaluation of the degree of GFP segregation in CA1 showed a positive correlation with seizure severity (Pearson correlation coefficient r(9)= .67, p=0.023).

Conclusions: Young PCE mice have a lower seizure threshold and more severe seizures when exposed to hyperthermia. Hyperthermic seizure severity positively correlated with the degree of GFP+ and GFP- cell segregation within the CA1 region of the hippocampus, suggesting that random X-inactivation patterns underlie the widely variable phenotypes observed in patients with PCE. Further studies are needed to better elucidate the underlying mechanisms responsible for PCE.

Funding: Please list any funding that was received in support of this abstract.: This work was supported by NIH-NICHD Child Health Research Center Development Award - 5K12HD028820 (PI: Martin).