Identification And Functional Analysis Of NEFH Gene In A Pedigree With Charcot-Marie-Tooth Disease

BackgroundCharcot-Marie-Tooth?CMT?disease is the most common type of inherited peripheral neuropathies with high clinical and genetic heterogeneity,the prevalence of which is 1/2500.Typically,it's characterized as symmetrical weakness and atrophy of distal limbs,with abnormal gait,crane leg deformity,pes cavus,hammer toes,and weakened or disappeared deep tendon reflex.Sensory loss occurred occasionally in patients.On the basis of electrophysiological features,CMT can be classified into two broad categories,demyelinating type?CMT1?,with significantly reduced motor nerve conduction velocity?MNCV<38 m/s?as well as demyelinating changes;axonal type?CMT2?,with preserved MNCV and axonal degeneration.Besides,patients with MNCV among 35 to 45 m/s are characterized as middle type.With the rapid development and widespread utilization of high-throughput sequencing,up to now,more than 70 causative loci have been identified responsible for CMT.The disease displays several distinct patterns of inheritance,including autosomal dominant inheritance?AD?,autosomal recessive inheritance?AR?and X-linked inheritance.CMT can be classified into various subtypes according to its inheritance patterns,and each subtype has its own clinical phenotype.In peripheral nervous system,Schwann cells play a pivotal role in the formation of myelin sheath and the maintenance of its structure stability.One pathological change of CMT is that mutations of some causative genes can lead to the production of misfolded proteins,which can abnormally accumulate in the endoplasmic reticulum and thus cause the unfolded protein response,with increased apoptosis of Schwann cells and demyelinating lesions.While providing nutrition support for neurons,Schwann cells have great significance in the maintenance of its structure and function,thus leading to accompanying damages in axons along with demyelinating changes.And compared to the demyelination alone,the appearance of subsequent axonal degeneration has closer connection with clinical phenotypes.Moreover,some causative genes can directly affect the development of axons,thus leading to axonal degeneration and the onset of CMT.Neurofilaments?NFs?,a kind of intermediate filament whose diameter is 10 nm,are classi:fied into three types according to the molecular weight,namely,nerofilament light chain?NEFL?,nerofilament medium chain?NEFM?,and nerofilament heavy chain?NEFH?.Neurofilament proteins are initially formed in neuronal cell bodies,and are subsequently transported to axon,where they constitute network structure with other cytoskeleton proteins,altogether functioning in axon growth,maintenance of axon stability,and transduction of electrical impulses along with axon.It's reported that mutations in NEFL can lead to abnormal aggregation of mutant preteins in neuronal cell bodies,disruption in formation of normal network structure,and thus causing dysfunction in axon transport,axonal degeneration,and as a result,the development of CMT2E.Mutations in NEFM were reported to be associated with Parkinson's disease?PD?.Furthermore,mutations in NEFH can contribute to Amyotrophic lateral sclerosis?ALS?,also a common type of degenerative neuropathies.Above all,the normal assembly of neurofilament network is of great significance in elongation and transportation of axon,so that mutations in genes that encoded proteins associated with neurafilaments often lead to development of neuronal disorders.Objectives1.To identify the causative gene of an AD-inherited CMT pedigree.2.To further investigate the pathogenic mechanism of the variant in vitro and vivo levels.Methods1.DNA underwent whole-genome linkage analysis for all family members and whole-exome sequencing for 2 affected members.2.Cell lines with stable expression of WT or mutant NEFH were constructed.Immunofluorescene was used to detect the distribution of NFs in the cells.3.The nefh knockdown zebrafish model was established using MOs.The morphology and motility of the embryos were observed.The axonal development of neurons was detected by immunohistochemistry and immunofluorescene.The pCS2-NEFH and pCS2-NEFH-Mut eukaryotic expression vectors were constructed and linearized.Human WT and mutant NEFH mRNA were obtained by transcription in vitro and coinjected with nefh MOs respectively.The phenotype of embryos was observed.4.Human mutant NEFH transgenic mice and mutant Nefh knock-in mice were established.Behavior was observed by rotarod test,treadmill test and grip strength test.The eletrophysiological characteristics of mice were examined,including nerve conduction velocity?NCV?and compound muscle action potential?CMAP?.The morphology of muscle fibers was observed by H&E staining.The sciatic nerve of the mutant Nefh knock-in mice was analyzed histologically by transmission electron microscope.Results1.Identification of NEFH gene in a pedigree with Charcot-Marie-Tooth diseaseWe collected a CMT pedigree with three generations,in which patients exist in each generation with both males and females,demonstrating the inheritance pattern for this family is autosomal dominant inheritance.Clinically,it's diagnosed as CMT2E.Altogether,up to now,it seemed that there are 7 affected individuals and another 10 normal members.The onset of the disease is around age 20,with classical phenotypes as weakness and atrophy in lower limbs,decreased or absent tendon reflex,and gradually involvement in upper limbs,meanwhile,other skeletal malformations,like talipes cavus,and without involvement in sensory function.These results can only prove that NEFH has an effect on motor neurons,but can not verify that NEFH?p.Lys1020Glufs*43?has pathogenic effect.In order to identify the pathological gene of this pedigree,after excluding the known causative genes of CMT,we conducted genome-wide scanning upon 17 members with Illumina single nucleotide polymorphism?SNP?chip and genotyped upon 872,261 SNPs in autosomal chromosomes.As a result,we identified two regions whose LOD scores were over 2,as a signal of linkage,171204750-173914775 in chromosome 2 with 2.3543,28866225-36418280 in chromosome 22 with 2.3541.Next,Haplotype analysis was carried out on these two candidate regions,confirming its co-segregation with phenotype.Meanwhile,we performed whole-exome sequencing in two patients with distant relationship??;?4?.Mutations which located in linkage regions and shared by both two patients were seemed as candidate mutations.As a result,we found no eligible mutation on chromosome 2,while 4 candidate mutations on chromosome 22,namely,NEFH?c.1937₁938insAAGTCCCCTGAGAAGGCC?,NEFH?c.2229₂248deIGCTAAGTCCCCAGAGAAG?,NEFH?c.3057insG?,and APOL6?c.824 C>A?,none of which exited in the 1000 genome project database.Among them,however,the first mutation in NEFH was excluded since a homozygous mutation cannot be the pathological reason for an AD-inherited pedigree;as a polymorphic site in Exome Aggregation Consortium?ExAC?database,the second mutation in NEFH was also excluded;and same destiny for the mutation in APOL6,which can be found in ExAC database.Above all,we regarded the third mutation in NEFH?c.3057insG?as the most plausible pathological gene.What's more,we performed Sanger sequencing over all members and confirmed its phenotype-genotype correlation.Therefore,we initially identified the causative gene for this pedigree as NEFH?c.3057insG?.NFs are mainly synthesized in neuronal cytoplasm,and are transported to distant regions along axon.In normal circumstances,in peripheral nervous system,NEFL,NEFM,NEFH and peripherins can assemble to form a network structure,which is of great significance for the maintenance of cytoskeletons and growth of axon.It's reported that,mutations in NEFL can lead to abnormal aggregation of neurofilaments in cytoplasm and subsequent disruption of normal network structure,thus causing development of CMT.Furthermore,mutations in NEFH can bring about aberrant amyloid aggregation in neuronal cell bodies,which is a classical pathological change for ALS.In order to further clarify the function and its pathogenic mechanism of NEFH mutation,we constructed viral vectors encoding wild-type?WT?or mutant type NEFH.Since SW13?vim-?cells don't express endogenous NFs,we chose SW13?vim-?cells to perform functional analysis of mutation and constructed SW13?vim-?cells over-expressing WT or mutant NEFH.NEFH must combine with NEFL to assemble NFs with a diameter of 10 nm,thus WT NEFL were transiently transfected into SW13?vim-?over-expressing WT-NEFH or MT-NEFH.After 48 hours,the expression of-NFs was detected by immunofluorescence with antibody of NEFH.It showed that the green fluorescence evenly dispersed in the cytoplasm in cells over-expressing WT NEFH while abnormal aggregation of green fluorescence was observed in the cells over-expressing mutant NEFH,indicating that the mutation can disrupt the formation of neurofilament network structure,and therefore lead to disease.2.Verification of mutant NEFH pathogenecity in zebrafishThe nervous system of zebrafish can be fully developed in a short period of time and is easy to be observed,thus making zebrafish as a model animal to study the pathological mechanisms of neurodegenerative diseases frequently.Therefore,we designed morpholino antisense oligonucleotides?MOs?aimed at the splicing site between the first intron and the second exon of nefh,and constructed nefh-knockdown zabrafish model with injecting MOs into embryos at 1-2 cell stage.Compared with embryos injected with control MOs that have 5 mismatched bases,we analysed their changes in anatomical and behavioral phenotypes.It showed that embryos with nefh MOs exhibited curled tail in varying degrees,while embryos with control MOs exhibited nearly as normal as WT embryos.Since the representative characteristics of CMT patients is symmetrical weakness and atrophy of lower limbs,we examined the motility of embryos with touch-response testing at 48 hours post fertilization?hpf?.In response to touch,nefh morphants were highly insensitive,who can only swim over short distance or completely lose capacity to react,while control morphants and WT embryos were extremely sensitive and reacted immediately with rapid swimming,indicating impaired motility is caused by knockdown of nejh.NFs play a pivotal role in the development of motor neuron axons,and that the typical pathological change of CMT2 patients is axonal degeneration.Thus,we examined the spinal motor neuron axons of embryos at 48 hpf with Znp-1,a specific marker for motor neuron axons.The immunolstaining results demonstrated aberrant axon pathfinding of spinal motor neuron axons with truncation and branching,and even agensis,while axons for control morphants and WT embryos can normally extend from dorsal side to ventral side.Besides,we obtained similar results by immunolabeling with acetylated?-tubulin.This indicated that the absent of nefh affected the normal growth of motor neuron axons and therefore led to motor disability.To further ascertain whether NEFH is the pathological gene of this CMT pedigree,we performed rescue experiments by co-injecting nefh MOs with human NEFH mRNA into embryos at 1-2 cell stage.We found that the tail deformities were obviously alleviated and the abnormal pathfinding phenotype of axons was rescued indicating the loss of endogenous nefh can be compensated by human NEFH mRNA.Mutant mRNA of NEFH,however,cannot rescue the aberrant phenotypes caused by loss of nefh,which further verified the pathogenicity of NEFH mutation.Besides,co-injection of nefh MOs and human NEFH mRNA can rescue aberrant phenotypes,whereas co-injection of nefh MOs and mutant NEFH mRNA cannot,which further demonstrated that the mutation in NEFH can lead to axonal degeneration and development of neurodegenerative disease.3.Pathogenic mechanism study of NEFH in mouse modelsTo further explore the function of this mutation,we constructed human mutant NEFH?mNEFH?transgenic mouse model.Initially,we observed changes in motility and muscular strength with rotarod test and grip strength meter.Compared with WT mice,however,no obvious lessening in athletic capacity and weakness in muscles were exhibited in mNEFH transgenic mice,which are typical characteristics for neurodegenerative disorders.Besides,the motor nerve conduction velocity?MNCV?and compound muscle action potential?CMAP?of transgenic mice showed no apparent changes.Furthermore,muscle analysis for transgenic mice showed well-arranged muscle fibers without muscle atrophy or other abnormal phenotypes.Above all,the mNEFH transgenic mice didn't appear analogous phenotypes as CMT patients or other similar animal models.Subsequently,we generated mNefh kncok-in mouse model.By using same methods,we observed no difference in motor ability and muscle strength in knock-in mice compared with WT ones in same age originally.Meanwhile,the MNCV and CMAP of knock-in mice both remained normal by electrophysiological test.Muscle analysis demonstrated normal array of muscle fibers without atrophy,hypertrophy or characteristic karyopyknosis.Electron microscopy detection of sciatic nerves of knock-in mice appeared decreasing axon numbers in contrast to WT mice,but normal axonal diameter,NF density and axon diameter/fiber diameter?g-ratio?.Although endogenous loss of nefh can lead to axonal degeneration of motor neurons in zebrafish,two kinds of mouse models appeared none apparent classic symptoms of neurodegenerative disorders.This is consistent with the absence of a significant clinical phenotype in the Nefh knockout mouse model,which may be due to the late onset of disease and the heterogeneity of the disease.SignificanceThe research found a new variant in NEFH associated with CMT2CC,which can cause abnormal accumulation of mutant proteins and disrupt formation of NFs network.The results further revealed the pathogenesis of CMT and provided new insights in genetic diagnosis.