Capture And Vertification Of Pathogenic Potassium Channel Gene And The Change Of Hippocampus MRS In Genetic Epilepsy With Febrile Seizures Plus

Genetic epilepsy with febrile seizures plus(GEFS+)is an epilepsy syndrome characterized by autosomal dominant inheritance with incomplete penetrance,and its genotype and phenotype are highly heterogeneous.Many pathogenic genes are still unknown,more unknown pathogenic and susceptible genes need to be explored further,so this study is committed to study the pathogenic gene of potassium channel in GEFS+ family.A high throughput gene capture approach was used to identify potassium channel gene which were then validated in functional verification,aimed to find the pathogenic gene in GEFS+.Then the change of hippocampus magnetic resonance spectrum(MRS)values in GEFS+ was discussed.Research objects and methodsGEFS+ families were included from January 2012 to December 2016 in pediatric epilepsy specialist outpatient and ward of Guangdong General Hospital,according to the International Antiepileptic Union(ILAE)2001 epilepsy and epilepsy syndromes diagnosis cretiria and GEFS+ definition.A total of 48 subjects,including 14 probands and their family members,were included for high throughput epilepsy target gene capture.A missense mutation of the KCNQ4 gene(P537S)was found in one GEFS +pedigree which was then validated in another 13 GEFS + families and 26 febrile seizures(FS)families in the same approach.Sequencing PCR products was used to screen gene mutations in 200 healthy subjects.KCNQ4P537S and KCNQ4WT were constructed in plasmid and then transfected into HEK293 cells with liposome.The mRNA levels of KCNQ4P537S and KCNQ4 wild type(WT)were compared by realtime fluorescence quantitative PCR(RTFQ PCR)and the protein level of KCNQ4P537S and KCNQ4WT were compared by Western blot.Potassium channel currents in Kv7.4 P537S homotetramer and Kv7.4 P537S/Kv7.3 WT heterotetramer channels were compared with those in Kv7.4 WT and Kv7.4 WT/Kv7.3 Wt by whole-cell patch clamp.14 cases of GEFS+ probands were included in hippocampus MRS research during the same period,the value of NAA/Cr?Cho/Cr?NAA/(Cho + Cr)of bilateral hippocampus were compared.Result1.In 14 GEFS+ families,a KCNQ4 gene mutation c.1609C>T was found in exon 12 which could cause the following protein variants:p.Pro537Ser(proline-serine).Two KCNQ4 gene mutations c.1177G>T,c.1207G>A were found in another FS families in exons 9,which could cause the following protein variants:p.Gly393Cys(glycine-cystine),p.Ala403Thr(alanine-threonine).Each patient only had one of these mutations among these three families.These sites have not been reported both at home and abroad.2.KCNQ4 gene mutations were not found in another GEFS+ families and the healthy control group.3.RTFQ PCR:The relative mRNA level of KCNQ4P537S was 0.776852 compared with that of KCNQ4WT,there was no statistical difference between them,p value was 0.1.4.Western blot:The relative protein level of KCNQ4P537S was significantly different from that of KCNQ4WT,p value was 0.0289.5.Patch clamp:Conventional whole cell mode recordings were generated from a holding potential of-90 mV to step potentials ranging from-70 to 60 mV using voltage increments of 10 mV.The channels of Kv7.4 P537S and Kv7.4 WT group,Kv7.4 P537S/Kv7.3 WT and Kv7.4 WT/Kv7.3 WT group were open at about-70mv and the channel current reached the peak value at about 50mV.The current density of P537S group was 13.27 ± 2.144 pA/pF(n = 10),and the current density of Kv7.4 WT was 25.8 ± 4.147 pA/pF(n = 8).The difference between them was statistically significant,p value was 0.0116.The I-V curve of the Kv7.4 P537S group was shifted to right compared with that in Kv7.4 WT group.The current density of Kv7.4 P537S/Kv7.3 WT was 32.15 ± 2.622 pA/pF(n = 8),and the current density of Kv7.4 WT/Kv7.3 WT was 61.69 ± 9.225 pA/pF(n = 10).The difference between them was statistically significant,p value was 0.0135.The I-V curve of Kv7.4 P537S/Kv7.3 group was shifted to right compared with that in Kv7.4 WT/Kv7.3 WT.The V50 of Kv7.4 WT t in activation curve was 14.3mv,while that of Kv7.4 P537S was 11.3mv,there was no statistical difference between them.Kv7.4 WT/Kv7.3 WT group V50 was 3.485mv,while Kv7.4 P537S/Kv7.3 WT was 9.955mv,the difference was statistically significant,and Kv7.4 P537S/Kv7.3 WT activation curve shifted to right.6.The mean value of NAA/(Cr + Cho)of the bilateral hippocampus MRS of the probands in GEFS+ was 0.71 ± 0.11,the normal control group was 0.74 ± 0.10 and there was no statistical difference between them,p value was 0.214.Conclusions1.A mutation of KCNQ4 gene c.1609C>T was found in one GEFS + family,and two mutations of KCNQ4(c.1177G>T?c.1207G>A)were found when validated in another 26 FS family.2.KCNQ4P537S affected the stability of the protein structure.3.The patch clamp results showed that KCNQ4P537S had an influence on the cell membrane potential,the current density decreased,the number of ion channels opened on the cell membrane was reduced,cell membrane repolarization process was prolonged,inhibition of neuronal excitatory activity was weakened,which might increase the neuronal excitability and promote the occurrence of epilepsy.4.KCNQ4 might be a new mutant gene for GEFS+ in Chinese population.5.Hippocampus fuctional magnetic resonance(MRS)in GEFS+ in this study had no changes.