A Novel Mutation In AFAP-120 Gene Causes An Autosomal-dominant Charcot-marie-tooth Disease Type 2

Charcot-Marie-Tooth(CMT)disease,also called Hereditary Motor and Sensory neuropathy(HMSN),is the most common inherited neurological disorder,affecting approximately 1 in 2,500 people.Some CMT patients develop severe disability in infancy or early childhood,while others experience symptoms of neuropathy until adulthood.CMT phenotype is characterized by distal weakness,progressive distal muscle atrophy,foot deformities(pes cavus and hammer toes),sensory loss,and so on.CMT is generally not fatal or life threatening,but the quality of patient life is affected severely.CMT is classified into three groups based on motor nerve conduction velocity(MNCV): demyelinating forms(CMT1,MNCV<38m/s),axonal forms(CMT2,MNCV>38m/s)and intermediate forms(ICMT,25<MNCV< 45 m/s).CMT is a clinically and genetically heterogeneous group of inherited diseases.The list of genes associated with CMT is continually growing.More than 80 genes have been described and about half of these CMT associated genes were discovered after 2009 due to the development of whole-exome sequencing.While more than 90% of patients with CMT1 achieve an accurate molecular diagnosis,25-43% of patients with CMT2 remain without a genetic diagnosis,even in the most recent clinical series.Identification of new CMT2 associated genes will contribute to establish the genetic cause of CMT2 and improve the diagnostic efficiency of CMT2,and help patients take accurate therapeutic methods.We found previously a family from Shandong Province with five affected individuals with an autosomal dominant pattern of inheritance.A detailed clinical diagnosis was carried out on patients from this family and the clinical characteristics were recorded.Patients of this family presented some CMT symptoms,such as distal limbs muscle atrophy,claw hands,pes cavus and foot drop.Electrophysiological studies showed a slight decrease in median and ulnar motor nerve conduction velocities(36-47 m/s),while the compound muscle action potentials showed dramaticly decreased amplitude in almost all nerves.Sural nerve biopsy showed pathological atrophy occurred in the axons of myelinated fibres and few layered myelin sheaths.Based on the evidences above,the inherited neurological disorder of the Shandong family was diagnosed as CMT2.A multi-gene test was conducted on the genome of patients,but did not find the genetic cause of this CMT2 family.To detect the gene associated with this CMT2 family,whole-exome sequencing was done and screened 82 candidate variants.Candidate variants were then confirmed with Sanger sequencing and segregation analysis.The heterozygous c.1621C>T change in the AFAP-120 gene,which was predicted to cause the amino acid substitution p.P541 S,was identified be the pathogenic mutation.The screening of c.1621C>T in healthy controls and 148 CMT2 patients did not show this genetic variant.The heterozygous c.1738A>G(p.N580D)change was discovered in a CMT2 pateint,which was then confirmed as a SNP.Human AFAP-120 gene is located on chromosome 4 at p16.1 and encodes an actin filament associated protein composed of 814 amino acids.AFAP-120 is an alternative splicing isoform of AFAP-110,containing an additional proline-rich neuronal insert(NINS)motif.The mutation P541 S is located exactly in NINS motif.While AFAP110 is expressed widely in most cell types,AFAP120 is specifically expressed in the nervous system.Compared to AFAP-110,relatively little is known about the function of AFAP-120,especially the function of NINS motif.To date,there is no reference about CMT2 associated with AFAP-120 variant.Sequence alignment analysis shows the proline at 541 site is highly conserved among species,which inplys that this proline may play a key role in the function of AFAP-120.The variant c.1621C>T(p.P541S)was predicted to be disease-causing by PolyPhen-2(with a score of 0.996 using HumVar model)and MutationTaster programs.Genetic and in silico studies strongly supported the pathogenicity of this variant.To evaluate whether the c.1621C>T mutation was sufficient for inducing the CMT2 phenotype and explore the pathophysiologic mechanism of this mutation leading to CMT2,we generated a transgenic mouse line expressing mutated AFAP-120(P542S)protein.Heterozygous AFAP-120P542 S mice were mated and siblings were used for comparative analyses between different genotypes.We tested the motor and sensory capacities of AFAP-120P542 S mice using rotarod test,footprint test or hot plate test.Female homozygous AFAP-120P542 S mice after 11 months of age showed a sensory loss and male homozygous AFAP-120P542 S mice showed a gait defect.Superficial peroneal nerve biopsy of two groups of mice with different genotypes were analysed using electron microscope.Micrographs of both heterozygous and homozygous AFAP-120P542 S mice showed many blank bulbs appeared in axonal compartment of myelinated fibres.The average area of axonal compartment decreased in both myelinated and unmyelinated fibres by about 30%.Superficial peroneal nerves of both heterozygous and homozygous AFAP-120P542 S mice in another group emerged aberrant thick myelin sheaths,and the average thickness increased by 0.080 and 0.167 ?m respectively.Axonal compartment atrophy appeared in both myelinated and unmyelinated fibres.The average area of axonal compartment decreased in myelinated fibres of heterozygous and homozygous AFAP-120P542 S mice by 0.520 and 4.429 ?m2 respectively.Some mitochondria in both myelinated and unmyelinated fibres swelled and experienced a deletion of cristae.To investigate the molecular mechanism of AFAP-120 c.1621C>T(p.P541S)leading to CMT2 and explore the functions of AFAP-120 and NINS motif,we performed co-immunoprecipitation assay using HEK293 T cells overexpressed AFAP-120(P541S)and immunofluorescence assay using SH-SY5 Y cells overexpressed AFAP-120(P541S).Results indicated that the interaction between AFAP-120(P541S)and ?-actin was strengthened and caused aberrant F-actin clusters in SH-SY5 Y cells.Actin filament is a major component of the cytoskeleton and plays crucial roles in morphology maintain of neuron and Schwann cells and in the process of myelin production.As an actin filament associated protein,mutated AFAP-120 may cause actin cytoskeleton dysfunction,and then leads to CMT2 disorder.In summary,we found a new form of axonal CMT disease and identified the pathogenic mutation AFAP-120 c.1621C>T(p.P541S).We generated AFAP-120P542 S transgenic mouse and found the pathological changes occurred in axons of peripheral neurons of transgenic mouse were same with those in CMT2 patient.We explored the molecular mechanism of AFAP-120(P541S)leading to CMT2 and found mutated AFAP-120 caused aberrations in actin filaments.Our studies add a new pathogenic mechanism to the long list of causes of CMT2 disease and can be very helpful for molecular diagnosis and genetic counseling for the CMT2 family from Shandong Provience and for related CMT2 diseases.In addition,this paper also shed light on the development of rational therapies for AFAP-120-related inherited neuropathies and the function research of AFAP-120 protein.