Clinical And Pathogenesis Studies Of KCNQ2 And KCNB1 Related Neurological Diseases In Children

Objective:Both variants in KCNQ2 and KCNB1 could cause pediatric neurological diseases.The study aimed to summarize the clinical characteristics and genotypes of these two disorders,and to evaluate the efficacies of antiseizure medications(ASMs)in patients with both diseases.To further establish the phenotype and genotype correlations of KCNQ2 and KCNB1 related diseases,patch clamp and western blotting(WB)were adopted to analyze the functional alterations of the variants detected from the enrolled patients.Methods:(1)Patients with pathogenic or likely pathogenic KCNQ2 variants and copy number variants(CNVs)containing KCNQ2 in Xiangya Hospital,Central South University(CSU)from July,2015 to January,2022 were enrolled in the study.They were classified into three subgroups:"developmental epileptic encephalopathy(DEE)","selflimited epilepsy",and "intermediate phenotype",according to their clinical manifestations.Comparisons of seizure attacks,electroencephalogram(EEG)features,ASMs efficacies,cognition developmental level and variant information were made among individuals in the three groups.Patch clamp was used to analyze the functional alterations of KCNQ2 missense variants with typical clinical phenotypes,such as variant p.S247L and variant p.P335L in DEE patients,variant p.D259G in a self-limited epilepsy patient,variant p.R75C,variant p.R144Q and variant p.P335A in patients with intermediate phenotype.(2)We collected the clinical and genetic information of patients with pathogenic or likely pathogenic KCNB1 variants in Xiangya Hospital,CSU from January,2016 to January,2021.Functional experiments including patch clamp and WB of the novel KCNB1 variants identified in our patients were performed in vitro.Literature review was conducted on Pubmed with the keywords of "KCNB1" or "Kv2.1" and "mutations" or"variants".To explore the possible correlations of molecular phenotypes and disease severity in KCNB1 related disorder,patients from our cohort and literature were classified into "Complete" or "Partial loss-of-function(LoF)" subgroup and "Dominant-negative" or "non dominant-negative effect" subgroup according to their KCNB1 variants’ functional consequences.Statistical analyses were made between "Complete LoF"and "Partial LoF" group,or "dominant-negative effect" and "non dominant negative effect" group.Results:(1)A total of 18 patients with KCNQ2 related DEE,22 with KCNQ2 related self-limited epilepsy,and 5 patients with intermediate phenotypes were included in the study.There were 18 DEE and 16 selflimited epilepsy patients with neonatal epilepsy,respectively.Compared with the "self-limited epilepsy" group,more individuals in "DEE" group experienced≥2 kinds of seizure forms(p<0.05),and required≥3 kinds of ASMs(p<0.05).Moreover,the initial EEG of DEE patients showed severe abnormal,and long-term EEG failed to turn into normal(p<0.05).The efficacies of sodium channel blocker(SCB)in self-limited epilepsy and DEE patients were better than that of non-SCB drugs(p<0.05),while the effective rate of phenobarbital in self-limited epilepsy patients was significantly higher than that in DEE group(p<0.05).The efficacies of sodium valproate in patients with self-limited epilepsy and DEE were 100%and 75%,respectively,while topiramate were 100%and 42.9%,respectively.Among the 5 patients with intermediate phenotype,3 patients were diagnosed as intellectual disability(ID)and epilepsy,and 1 patient was diagnosed with non-syndromic intellectual disability(NS-ID).All 3 patients with ID and epilepsy achieved seizure-free.KCNQ2 missense variants were detected in 40 patients,frameshift variants and splicing variants in 2 self-limited epilepsy patients respectively,and CNVs duplication containing KCNQ2 found in a patient with ID and epilepsy.Negligible or no current was recorded in cells expressing homomeric p.S247L or p.P335L which were found in DEE patients,suggesting they were severe LoF variants.The p.S247L failed to assemble with Kv7.3 subunits to generate larger Kv7 currents,while p.P335L presented marked dominant-negative behaviors with larger currents and hyperpolarized steady-state activation curves in heteromeric expression models.Variant p.D259G related to self-limited epilepsy had 25%～50%Kv7.2 subunit currents in the homomeric model,indicating it was a mild LoF variant.Moreover,p.D259G negatively regulated the currents of Kv7.2 subunits and led to a polarization shift of steady-state activation curve of Kv7.3 subunits.The variant p.P335A associated with ID and epilepsy exhibited a similar functional alteration to p.P335L in both homomeric and heteromeric models,but had a smaller effect on Kv7.2 and Kv7.3 subunits than the latter.In homomeric models,variant p.R75C in a NS-ID patient expressed larger outward currents and more hyperpolarized V50(about lOmV)than Kv7.2 subunits,while variant p.R144Q in a ID and epilepsy patient showed similar currents to Kv7.2 subunits and remarkably hyperpolarized V50(about 20mV).The above results supported both variants were gain-of-function(GoF)variants.When coexpressed with Kv7.2 or Kv7.3 subunits,p.R75C was almost"rescued",while p.R144Q was partially ameliorated.(2)A total of 10 patients with KCNB1 related disorder were reported in the study.Of these,9 had epilepsy,5 developed refractory epilepsy.Five of the 9 cases were treated with levetiracetam as the initial ASM,which was effective for 4 cases.One of the two cases with infantile spasms was responsive to ACTH.Two of the three cases that tried ketogenic diet had significant seizure reductions.All patients had developmental delays,and only 1 patient had normal cognition development at the last follow-up.Seven patients had behavioral problems,including autistic-like behaviors in 4,hyperactive in 3 and aggressive behaviors in 3.Nine different KCNB1 variants were detected,including 8 missense variants and 1 frameshift variant.Functional experiments of 8 novel KCNB1 variants revealed 3 partial LoF variants,including p.P272S,p.Q318H and p.E330D,and 5 complete LoF variants,including p.A192Pfs*1,p.S314P,p.T377I,p.G379V and p.P408S;6 dominant negative effect variants,including p.P272S,p.Q318H,p.E330D,p.T377I,p.G379V and p.P408S and 2 non dominant-negative effect variants,including p.A192Pfs*1 and p.S314P.33 patients(7 in the cohort,26 from the literature)and 13 patients(2 in the cohort,11 from the literature)were included in the "complete" and"partial LoF" groups,respectively;29 cases(5 in the study and 24 from the literature)and 17 cases(4 in the study and 13 from the literature)were included in the dominant-negative and non dominant-negative effect groups,respectively.Compared with those in the "Partial LoF" group,more individuals in the "Complete LoF" group had seizures within one year-old-age(64.3%vs 16.7%,p<0.05),and fewer individuals achieved seizure freedom(18.8%vs 66.7%,p<0.05).Patients in the "dominantnegative effect" and "non dominant-negative effect" group had similar seizure rates,seizure onset age,and seizure control rates.There were no significant differences in cognitive development and behavioral problems between "Complete" and "Partial LoF" group,as well as between"dominant-negative" and "non dominant-negative effect" group.Conclusion:(1)KCNQ2 related disorder has a wild spectrum of clinical phenotype.Besides KCNQ2 related DEE and KCNQ2 related self-limited epilepsy,ID with or without epilepsy is the most common intermediate phenotype.Neonatal onset seizure is an important clinical manifestation of KCNQ2 related disorder.The abnormal degree of early EEG and the treatment response of phenobarbital are key ingredients to predict prognosis of those patients.The epilepsy of KCNQ2-related DEE patients is the most severe and has the worst prognoses.The recommended first-line ASMs for these patients are SCB,and valproate has a higher priority than topiramate in add-on therapy.In contrast,patients with KCNB1 related disorders have distinct clinical features,including variable seizures,ID,and behavioral problems.Levetiracetam is effective ASM for KCNB1 related epilepsy,and ketogenic diet might be helpful for those with KCNB1 related refractory epilepsy.(2)Disease related KCNQ2 and KCNB1 variants are mainly missense variants,whereas truncating variants often associate with milder clinical phenotypes.(3)KCNQ2 missense variants could manifest as two molecular phenotypes:LoF variants and GoF variants.Severe LoF KCNQ2 missense variants relate with DEE,and mild LoF variants associate with SE.Whether KCNQ2 missense variants in DEE patients can be "rescue"by Kv7.2/Kv7.3 subunits correlates with their seizure severity.KCNQ2 GoF variants are associated with ID.In contrast,KCNB1 variants often are LoF variants,some patients with partial LoF KCNB1 variants tend to have milder epilepsy phenotypes.KCNB1 truncating variants may have a similar pathogenesis as KCNQ2 truncating variants,which cause disease through a haploinsufficiency mechanism.