Capture Of Novel Pathogenic Genes In Genetic Epilepsy With Febrile Seizures Plus And Functional Verification Of Potassium Channel Gene KCNT1

Genetic epilepsy with febrile seizures plus(GEFS+)is a type of genetic epilepsy syndrome with phenotypic and genotypic heterogeneity.Although GEFS+is known to be associated with genetic etiology,the etiology of many GEFS+families remains unknown and needs be further explored.Based on the previous study,we further expanded the GEFS+family for genetic screening.We found a new KCNT1 variant R706Q in a GEFS+family.Evaluation of gene pathogenicity indicated that the effect of this mutation on protein function was likely pathogenic.We speculated that KCNT1 variants might be the cause of GEFS+.The study intends to verify the KCNT1 R706Q variant using cellular functional method,and provides new theoretical basis for the etiology and precise treatment of GEFS+.Objects and methodsGEFS+families were included from October 2012 to October 2019 in Guangdong Provincial People’s Hospital.A total of 40 GEFS+families in the research group were collected.Genetic analysis was carried out by whole-exome sequencing technology using next-generation sequencing to the probands.The KCNT1 variants were verified by Sanger sequencing technology in 200 healthy people.The plasmid for KCNT1 R706Q and KCNT1 Wild-type(Wt)were constructed and packed with lentivirus.Then the lentivirus packaging plasmid for KCNT1 R706Q and KCNT1 Wt were transfected into HEK293 cells.Using quantitative RT-PCR,the relative mRNA levels of KCNT1 R706Q and KCNT1 Wt were tested.The protein level of KCNT1 R706Q and KCNT1 Wt were tested by Western blot.Potassium channels in KCNT1 R706Q and KCNT1 Wt were constructed.Both the two channels were intervened with 100 μM quinidine.The difference in the time for KCNT1R706Q channel and KCNT1 Wt channel to reach 50%of peak currents before quinidine intervention was tested by whole-cell patch clamp.The difference in current density between the two groups before and after quinidine intervention was also compared by whole-cell patch clamp.Results1.Among the 40 GEFS+families,21 families were found with pathogenic or likely pathogenic gene variants,including variants in SCN1 A,SCN3A,SCN9A KCNQ4,KCNAB3,KCNT1,GABRD and GPR98 gene.Gene variants with unknown significance were found in 3 families,including KCNQ3 variant,CACNA1A variant and CACNA1H variant.2.Novel variant R706Q and R1137H in KCNT1 gene were identified,which has not been reported according to the literature.The variant was not found in 200 healthy population.3.RT-PCR:Both lentivirus of KCNT1R706Q and KCNT1 Wt were effectively expressed at relative mRNA level.4.Western blot:Both lentivirus of KCNT1R706Q and KCNT1 Wt were effectively expressed at relative protein level.5.Patch clamp:The channels of KCNT1 Wt and KCNT1R706Q were open at about-80mv and the channel current reached the peak value at about 60mV.In the step stimulation,the current density of KCNT1R706Q group(58.42±18.28 PA/pF)was higher than that of KCNT1Wt group(39.96±6.31 PA/pF).There was no significant difference between the two groups(P=0.2575).The time for KCNT1 R706Q channel to reach 50%of peak current(0.1096±0.0066s)was less than the time for KCNT1Wt(0.1566±0.0059s).The difference was statistically significant(p=0.001).The time-voltage curves showed that the curves in KCNT1 R706Q group shifted to the right compared with that in KCNT1R706Q group.After 100 μM of quinidine intervention,the current density of KCNT1Wt group(10.75±1.71 pA/pF)was significantly decreased compared with that before quinidine intervention(P<0.0001),and so did the KCNT1 R706Q group(P<0.0001).As the voltage was up to 60mV,the current density after being intervened by quinidine(29.67±5.65 pA/pF)was significantly decreased in KCNT1 Wt group compared with that before being intervened by quinidine(154.34±24.64 pA/pF).The difference between of them was statistically significant(P<0.0001).In the KCNT1 R706Q group,the current density after quinidine intervention(37.74±7.22 pA/pF)was significantly lower than that before quinidine intervention(249.26±68.99 pA/pF)as the voltage was up to 60mV.The difference between them was statistically significant(P=0.0008).Conclusions1.The new variation sites in SCN1A gene(nine families),SCN9A gene(one family)and GABRD gene(one family)were found in 40 GEFS+families.2.The novel heterozygous missense variants in KCNT1 gene,namely R706Q and R1137H,were identified in 2 GEFS+families.3.The R706Q variant in KCNT1 gene caused functional change and resulted in channels which display a gain-of-function phenotype.KCNT1 might be a new disease-causing gene of GEFS+in Chinese population.