Clinical Characteristics And Pathogenic Mechanism Of Hypomyelinating Leukodystrophy Caused By TUBB4A Gene Variants

Objective: To summarize the clinical phenotypic characteristics of4 cases of hypomyelinating leukodystrophies(HLDs)caused by TUBB4 A gene variants.We conduct functional experiments in in vitro to investigate the pathogenic mechanism caused by TUBB4 A gene variants and further explore the pathogenic mechanism of TUBB4 A gene variants associated with different clinical phenotypes.Methods:(1)The clinical data of 4 children with TUBB4 A gene variants and their family members treated in the outpatient and inpatient departments of Xiangya Hospital were collected,including medical history,clinical manifestations,neuropsychological assessment,genetic test results,neuroimaging and video electrograms.We analyzed the clinical data,neuroimaging features and genetic characteristics.(2)Real-time quantitative PCR was performed in peripheral blood lymphocytes derived from 3 patients with TUBB4 A variants and 3normal controls to detect the changes of TUBB4 A gene transcription level.(3)Construction expressing human wild-type TUBB4 A and variant TUBB4 A associated with different clinical phenotypes.The p.R2 G variant results in dystonia type 4,the p.D249 N variant leads to hypomyelinating leukodystrophy with basal ganglia and cerebellar atrophy.The p.E410 K,p.Q292 K variants are linked to isolated hypomyelinating leukodystrophy,and the novel TUBB4 A variant p.K324 T is identified by our team in a patient characterized by clinacal hypomyelinating leukodystrophy with infantile epilepsy with mitigating focal seizures.(4)Wild-type TUBB4 A and variant TUBB4 A plasmids are overexpressed in HEK293 cells or He La cells.Then we analyze the effects of variant TUBB4 A on expression of protein TUBB4 A,the stability of microtubules,the sensitivity of microtubules to paclitaxel,the dynamic stability of microtubules and the binding ability of microtubules to kinesin by western blotting,cellular immunofluorescence,paclitaxel sensitivity detection,dynamic microtubule changes in living cells,and co-immunoprecipitation.(5)The primary cortical neurons of embryonic mice are cultured in vitro,and the primary neurons are transfected with the TUBB4 A plasmids to observe whether the variant TUBB4 A defect the development of neurons.Results:(1)The heterozygous TUBB4 A missense variants carried by the 4 patients in this study are all de novo.Three of the four TUBB4 A variants in three cases are previously reported(p.E410 K,p.Q292 K and p.V255I),present prominently movement disorders,progressive spastic paraplegia,and extrapyramidal symptoms(such as dystonia and ataxia),and intellectual disability.The case 4 with the novel TUBB4 A variant p.K324 T shows a new clinical phenotype,known as infantile epilepsy with migratory focal seizures,which is a kind of severe early infantile epilepsy encephalopathy with poor prognosis.The clinical features include infantile epilepsy with migratory focal seizures,delay in language and motor,intellectual disability,progressive spastic paraplegia and dystonia.All 4 patients show varying degrees of hypomyelinating leukodystrophy in brain neuroimaging,with or without cerebellar atrophy,without basal ganglia atrophy or loss.The atrophy or absence of the basal ganglia is the main diagnostic criteria and clinical neuroimaging features of hypomyelinating leukodystrophy with typical basal ganglia and cerebellar atrophy.Although the cases in this study don’t show obvious atrophy and deletion of the putamen and caudate nucleus,all 4 cases have clinical extrapyramidal features which suggests that these cases may have functional impairment in the basal ganglia.(2)In the lymphocytes of peripheral blood derived from our 3 patients,the m RNA expressions of TUBB4 A gene are significantly lower than that of the normal controls.(3)TUBB4A gene variants does not affect the binding of β-4A tubulin to endogenous α-tubulin,and the variant TUBB4 A protein can normally participate in the formation of microtubule network without affecting its subcellular localization.(4)The results of cell experiments in vitro show that TUBB4 A variants(p.D249 N,p.E410 K,p.Q292 K,p.K324T)increase the stability of microtubules.In addition,the proportion of insoluble microtubules in cells expressing the wild-type TUBB4 A and TUBB4 A variants(p.D249 N,p.Q292K)increased by paclitaxel.However,compared with wild-type TUBB4 A,the variants(p.D249 N,p.Q292K)show lower increase in the proportion of insoluble microtubules,while TUBB4 A variants(p.R2 G,p.K324 T and p.E410K)are not responsive to paclitaxel.These results suggest that all five TUBB4 A variants have reduced sensitivity to paclitaxel.(5)Compared with He La cells overexpressing wild-type TUBB4 A,the polymerization rate of microtubules in cells overexpressing variants p.D249 N,p.E410 K,p.Q292 K,p.K324 T decreased significantly,while in the cells overexpressing the variant p.R2 G the rate is similar to wild type.The data indicate that TUBB4 A variants p.D249 N,p.E410 K,p.Q292 K,p.K324 T impair the dynamic stability of microtubules,while the variant p.R2 G don’t.(6)In HEK293 cells,overexpressed TUBB4 A variants(p.K324 T,p.E410K)defect the binding ability of β-4A tubulin to endogenous kinesin KIF1 A.The other three TUBB4 A variants(p.R2 G,p.D249 N,p.Q292K)have no apparent effect on the binding of microtubules to the kinesin KIF1 A.(7)Accept the variant p.R2 G,the other four TUBB4 A variants all inhibit the growth of axons and neurites,reduce the number of neuronal dendritic spines,and increase the proportion of immature dendritic spines(thin dendritic spines).The data show that the TUBB4 A variants inhibit development of neurons.Conclusion:(1)This study report 4 cases of hypomyelinating leukodystrophy caused by TUBB4 A variants,without the atrophy of basal ganglia associated with typical H-ABC on brain MRI.One case is firstly associated with EIMFS phenotype,expanding the TUBB4 A genotype and phenotype.(2)We find that the m RNA expressions of TUBB4 A decrease in the peripheral blood lymphocytes of patients,and the loss of function is probably one of the pathogenic mechanisms of TUBB4 A variants.(3)Inhibited polymerization induced by paclitaxel is a common pathogenic mechanism of TUBB4A-related disease spectrum.Altered microtubule stability and microtubule dynamic stability,and abnormal growth and development of neuronal axons and dendritic spines may be the common pathogenic mechanisms of H-ABC and isolated hypomyelinating leukodysplasia,rather than the pathogenic mechanism of dystonia type 4.(4)The decreased binding ability of microtubules to kinesin is more common in isolated hypomyelinating dystrophy phenotypes.The effects of TUBB4 A variants on microtubules binding to kinesin might lead to different clinical phenotypes caused by TUBB4 A variants.Our study provides new insight on the pathogenic mechanism of different TUBB4 A variants associated with distinct clinical phenotypes.