Identification Of Liang-Wang Syndrome And Its Disease-causing Gene KCNMA1

The KCNMA1 gene encodes a large conductance and Ca²⁺-sensitive potassium channel（refered to as BK channel）.The BK channel is activated by both membrane depolarization and intracellular Ca²⁺.BK channels are highly expressed in the nervous system and smooth muscle cells,and involved in neurotransmitter release and smooth muscle contraction and relaxation.In 2005,the gain-of-function（GOF）mutation p.D434G of KCNMA1 was first found to be associated with coexistent generalized epilepsy and paroxysmal dyskinesia（GEPD）.Subsequently,another KCNMA1 GOF mutation p.N995S was identified and associated with epilepsy only.Currently,detailed studies on pathogenic variants and patient phenotypes of KCNMA1 are still lacking.There was an urgent need to identify more KCNMA1 mutations,explore its pathogenic mechanism,and elucidate the relationship between genotype（mutations）and clinical phenotypes of the patients（i.e.,genotype-phenotypes correlation analysis）.This dissertation focused on the study of KCNMA1 gene mutations and human diseases.We performed genetic analysis of multiple collected patients,screened for pathogenic mutations,identified pathogenic mechanisms,and elucidated the relationship between pathogenic mutations and clinical phenotypes.This dissertation includes two major parts:The first part is on,identification and characterization of Liang-Wang syndrome.We analyzed the phenotypes of 11 patients with developmental delay,intellectual disability,ataxia,and other dyskinesia,and their family menmbers.The clinical phenotypes of the patients include global developmental delay,intellectual disability,speech delay,ataxia,seizures,and facial and visceral malformations（including hypertelorism,broad nasal root,gingival hyperplasia,gastrointestinal atresia and aortic dilatation,skeletal dysplasia）.Whole exome/whole genome sequencing in combination with bioinformatic analysis identified ten mutations in the KCNMA1 gene.Mutations p.A172T,p.A314T,p.S351Y,p.G356R,p.G375R,p.I663V,p.P805L and p.D984N were de novo mutations as only the probands carry the respective mutations but their parents did not carry these mutations.Further bioinformatic analysis showed that these ten mutations were very harmful and deleterious,had a low frequency in the common databases,and occurred at highly conserved amino acid sequences of the BK channel among different species.Electrophysiological results showed that mutations p.A172T,p.S351Y,p.G356R,p.G375R,p.N449fs and p.I663V completely blocked BK channel current.Mutations p.A314T,p.C413Y and p.P805L reduced both BK channel current and sensitivity to the voltage-dependent activation.The p.D984N mutation reduced the BK current without affecting activation kinetics.Altogether,our data indicate that mutations p.A172T,p.A314T,p.S351Y,p.G356R,p.G375R,p.C413Y/p.N449fs,p.I663V,p.P805L and p.D984N mutations are all loss-of-function（LOF）mutations.Furthermore,the p.A172T and p.A314T mutations inhibited BK channel currents when complexed with the wild type BK channel;the p.A172T mutation inhibited the Mg²⁺sensitivity.Biochemical experiments showed that p.A172T and p.A314T mutant subunits can interact with wild type subunits to form heteromeric channels.Altogether,we identified and characterized 11patients who showed a new spectrum of developmental and neurological phenotypes,and carried mutations that caused disease through the LOF mechanism.The Online Mendalian Inheritance of Man（OMIM）named this rare disease as“Liang-Wang Syndrome”.This study established the causal relationship between LOF mutations of the KCNAM1 gene and Liang-Wang syndrome.The second part is on,identification and molecular characterization of KCNMA1variants associated with GEPD.We identified two new patients and their family members.Interestingly,the new patients showed intellectual disability,developmental delay,mild cognitive impairment and mild dysmorphic features,in addition to similar features of epilepsy and dyskinesia as patients with GEPD.Whole exome sequencing and bioinformatic analysis identified two de novo mutations in the KCNMA1 gene,p.N536H and p.N995S.These two mutations were reported as GOF mutations,and this study further characterized the two mutations,in particular,at the heterozygous state mimicking the human pateints.Electrophysiological experiments showed that p.N536H and p.N995S mutants increased BK channel current and sensitivity to the voltage-dependent activation even when complexed with wild type channels.Moreover,the p.N536H mutation also reduced Ca²⁺sensitivity of BK channels.Biochemical experiments showed that p.N536H and p.N995S mutant subunits can interacted with wild type subunits to form heteromeric channels.Therefore,p.N536H and p.N995S mutations are KCNMA1 GOF mutations.In summary,we identified and characterized Liang-Wang syndrome and multiple LOF mutations of the KCNMA1 gene for the first time,established the causal relationship between the KCNMA1 LOF mutations and Liang-Wang syndrome.Furthermore,two new patients with GEPD presented novel clinical features that expanded the clinical phenotypes of KCNMA1 GOF mutations.Therefore,this study provided a framework to develop strategies for the prevention,screening and diagnosis of patients with developmental delay,intellectual disability,ataxia,and other dyskinesia.