1
A TFG mutation causes autosomal dominant axonal
Charcot-Marie-Tooth disease
Pei-Chien Tsai,1,2,3,13 Yen-Hua Huang,4,5,13 Yuh-Cherng Guo,6,7 Hung-Ta Wu,8
Kon-Ping Lin,1,2 Yi-Chu Liao,1,2 Cheng-Tsung Hsiao,1,2 Yi-Chun Lu,1,2 Tze-Tze Liu,9
Shaw-Fang Yet,10 Ming-Ji Fann,3,11 Lun-Sen Kao,3,9,11 Bing-Wen Soong,1,2,3,12,*
Yi-Chung Lee1,2,3,*
1
Department of Neurology, Taipei Veterans General Hospital, Taipei 11217, Taiwan;
2
Department of Neurology, National Yang-Ming University School of Medicine,
Taipei 11221, Taiwan; 3 Brain Research Center, National Yang-Ming University,
Taipei 11221, Taiwan; 4Department of Biochemistry, National Yang-Ming University
School of Medicine, Taipei 11221, Taiwan; 5Center for Systems and Synthetic
Biology, National Yang-Ming University, Taipei 11221, Taiwan; 6Department of
Neurology, China Medical University Hospital, Taichung 40447, Taiwan; 7Instute of
Clinical Medicine, National Yang-Ming University School of Medicine, Taipei 11221,
Taiwan; 8Department of Radiology, Taipei Veterans General Hospital, Taipei 11217,
Taiwan; 9Department of Life Sciences and Institute of Genome Sciences, National
Yang-Ming University, Taipei 11221, Taiwan; 10Institute of Cellular and System
Medicine, National Health Research Institues, Zhunan 35053, Taiwan; 11Genome
Research Center, National Yang-Ming University, Taipei 11221, Taiwan;12Institute of
Neuroscience, National Yang-Ming University, Taipei 11221, Taiwan; 13These
authors contributed equally to this work.
*Correspondence: ycli@vghtpe.gov.tw (Y.-C.L.) and bwsoong@ym.edu.tw (B.-W.S.)
2
Words in the main body: 3520
Abstract words count: 219
Short title: A TFG mutation causes AD-CMT2
3
Abstract
Charcot-Marie-Tooth disease (CMT) is a group of inherited neuropathies sharing
common features of progressive distal muscle atrophy and weakness, sensory loss,
foot deformities and depressed tendon reflexes. Mutations in approximately 60 genes
have been identified as being associated with CMT. Nevertheless, the genetic
etiologies of at least 30% of CMTs have yet to be elucidated. We studied a
five-generation pedigree with the autosomal dominant axonal CMT (CMT2) and
excluded mutations in common CMT2 implicated-genes by Sanger sequencing.
Subsequent exome sequencing of two affected individuals revealed a novel
heterozygous mutation, c.806G>T (p.Gly269Val), in the TRK-fused gene (TFG) that
perfectly co-segregates with the CMT2 phenotype in all 27 family members. This
mutation occurs at an evolutionarily conserved residue and is absent in the 1,140
ethnically matched control chromosomes. Cell transfection studies showed that the
TFG p.Gly269Val mutation increased the propensity of TFG proteins to form
aggregates, which sequestrated both mutant and wild-type TFG and might deplete
functional TFG molecules. The secreted Gaussia luciferase reporter assay
demonstrated that inhibition of endogenous TFG resulting in compromised protein
secretion pathways, which could be rescued by expressing wild-type TFG but not the
p.Gly269Val altered proteins. This study identifies a novel cause of CMT2 and
highlights the importance of TFG in protein secretory pathways which are essential
for proper functioning of human peripheral nervous system.
Keywords: Charcot-Marie-Tooth disease, CMT, exome sequencing, TFG, TRK-fused
gene, CMT2.
4
Charcot-Marie-Tooth disease (CMT), also called Hereditary Motor and Sensory
Neuropathy (HMSN), is a clinically and genetically heterogeneous group of inherited
neuropathy sharing common features of progressive distal muscle atrophy and
weakness, distal sensory loss, foot deformities and depressed tendon reflexes. It is one
of the most common inherited neurological disorders with a prevalence of about
1/2500. CMT can be classified into a demyelinating or axonal subtype according to its
electrophysiological or pathological features. Usually, in nerve conduction studies,
demyelinating CMT (CMT1) is associated with significantly reduced nerve
conduction velocities (NCVs), whereas axonal CMT (CMT2) is accompanied with
reduced amplitudes and relatively preserved NCVs.1-3 Clinically, median motor NCV
is the most commonly used parameter, with a cut-off value of 38 m/s to separate
CMT1 from CMT2.4 Mutations in more than 60 genes have been identified in CMT to
date and at least 15 of them are known for typical dominant CMT2, but the genetic
causes of more than 60% of CMT2 remain elusive.1-3
We studied a large Taiwanese family of Han Chinese origin and enrolled 27
members, including 16 individuals affected with autosomal-dominant CMT2, nine
unaffected individuals, and two married-in spouses (Figure 1A). Disease status was
ascertained by neurological examination and electrophysiological evaluation. The age
at disease onset ranged from 28 to 40 years. All affected individuals presented with
typical CMT2 phenotypes, including slowly progressive symmetrical muscle atrophy
and weakness from distal parts of limbs and mild distal sensory loss. The clinical
severity varied from being asymptomatic to being dependent on the assistance of a
walker for ambulation. The median motor NCVs ranged from 43.6 to 60.8 m/s.
Clinical manifestations of part of the affected individuals are shown in Table 1.
Written informed consents were obtained from all subjects and the study was
5
approved by the Institutional Review Board of Taipei Veterans General Hospital,
Taiwan. Genomic DNA was isolated from peripheral blood leukocytes following a
standard protocol.
The clinical features of the proband (IV-2) are used to examplify the disease in
this family. IV-2 is a 36-year-old man who had manifested slowly progressive
weakness of distal lower limbs since the age of 28 years. He started to walk at a
normal age. Physical examination at age 36 revealed high-arched feet, hammer toes,
generalized areflexia, atrophy and weakness of the intrinsic muscles of the hands
(score 4/5 in Medical Research Council Scale), feet (2/5) and also the muscles in the
legs, resulting in impaired dorsiflexion and plantar flexion of the feet (4 and 4+/5).
Despite a lack of sensory complaints, mildly diminished sensation for all modalities in
regions distal to the ankles was observed. Nerve conduction studies demonstrated the
presence of an axonal sensorimotor polyneuropathy with normal NCVs but reduced
amplitudes. A sural nerve biopsy revealed loss of large myelinated nerve fibers (MFs)
with relatively preserved median and small MFs (fiber density: 4366/mm2; fibers with
diameters larger than 7 um: 23.7%) and no signs of demyelination (Figure 1B and 1C).
Sanger sequencing excluded mutations in the majority of the genes known to be
putatively involved in CMT2;5 MFN2 (MIM 608507), RAB7 (MIM 602298), TRPV4
(MIM 605427), GARS (MIM 600287), NEFL (MIM 162280), HSPB1 (MIM 602195),
MPZ (MIM 159440), GDAP1 (MIM 606598), HSPB8 (MIM 608014), DNM2 (MIM
602378), and AARS (MIM 601065).
To identify the genetic cause of the CMT2 in this family, we employed a
whole-exome sequencing strategy as previously described.6 In brief, exonic regions
from the genomic DNA of the two affected individuals of the family (III-8 and IV-3 in
Figure 1A) were captured and enriched using the Agilent SureSelect Human All Exon
6
v2 kit. Then, enriched DNA was sequenced on an Illumina Genome Analyzer GAII
with 100 bp paired-end reads after standard sample preparation. Overall, an average
of 30.8 Gb sequence was generated per individual, resulting in an average coverage
depth of 398.1x per targeted base. After analysis and filtering (Table S1), 109
heterozygous coding variants were found to be shared by both patients and were not
present in the dbSNP, the 1000 Genomes Project,7 or the in-house exome data from 10
non-CMT ethnic controls. Sanger sequencing of these variants in other 25 members of
the family revealed that only one variant, c.806G>T (p.Gly269Val) in the TRK-fused
gene (TFG; MIM 602498) (RefSeq accession number NM_006070.5), perfectly
segregated with the CMT phenotype (Figure 1A and 1D). The pathogenic role of the
mutation, TFG p.Gly269Val, is further supported by its absence in the 1,140
ethnically matched control chromosomes and 6500 exomes in the Exome Variant
Server of the NHLBI Exome Sequencing Project, involvement of a highly conserved
residue (Figure S1) and being pathogenic as predicted by four different programs:
SNPs&GO,8 Panther,9 PMut10 and SNAP.11 Mutational analyses of TFG were further
conducted in 21 unrelated individuals with molecularly unassigned CMT2 and no new
family was found. Since the CMT2 individuals in this study were selected from a
continuous series of 251 CMT families after an extensive mutational analysis and
electrophysiological evaluation for CMT,5 the frequency of TFG mutation in our CMT
population was approximately 0.4% (1/251).
TFG was initially identified as a part of the neurotrophic tyrosine kinase receptor
type 1 (NTRK1)-derived thyroid oncogene, TRK-T3, whose protein product is a
chimera consisting of the N-terminal portion of the protein TFG and the C-terminus
of the tyrosine kinase NTRK1.12 Subsequent studies found additional TFG fusion
oncogenes in other human malignancies.13,14 TFG is ubiquitously expressed in normal
7
human tissues and localizes to endoplasmic reticulum (ER) exit sites in an oligomeric
form.15,16 It interacts with Sec16B and has an important role in the COPII-mediated
export of secretory cargo from the ER.16 Inhibition of TFG in cell lines compromises
the protein secretion from ER and alters the ER structure.17 Two mutations in TFG
were recently implicated in two neurological diseases, including a homozygous
p.Arg106Cys mutation in complicated hereditary spastic paraplegia and,17
interestingly, a heterozygous p.Pro285Leu mutation in hereditary motor and sensory
neuropathy with proximal dominant involvement (HMSN-P; MIM 60448).15
HMSN-P is an adult-onset autosomal dominant neuropathy characterized by proximal
predominant muscle weakness and atrophy, widespread fasciculations, muscle cramps,
and late onset of distal sensory involvement. In contrast to the typical clinical
manifestations of CMT presenting with predominantly distal muscle atrophy and
weakness, the clinical presentations of HMSN-P are more similar to those of
amyotrophic lateral sclerosis.15 The TFG p.Pro285Leu mutation has been reported in
four Japanese families and one Korean family with HMSN-P.15,18
To affirm that the phenotype of the affected individuals harboring the TFG
p.Gly269Val mutation in this family is typical CMT rather than HMSN-P, we utilized
skeletal muscle magnetic resonance imaging (MRI) to scrutinize the distribution of
their lower limb muscle atrophy. Two affected individuals (IV-2, IV-16) and one
healthy control subject were studied with MRI of the hip and lower limbs by using a
1.5-T MR unit (Signa CV/I, GE Medical Systems, Milwaukee, WI). T1-weighted
images [517-613 ms repetition time (TR), 7.98-8.34 ms echo time (TE), 10 mm
thickness] were used to look for the presence of muscle atrophy with fatty substitution.
Both affected individuals had a distally predominant muscle atrophy and fat
replacement with a proximal-to-distal gradient of muscle involvement on
8
T1-weighted MRIs (Figure 2), compatible with the features of typical CMT.
Individual IV-16, who was only mildly affected, had a mild fatty infiltration of
muscles in the legs (mainly gastrocnemius and soleus muscles) and a minimal
involvement of muscles in the thighs (mainly vastus lateralis muscle) (Figure 2B).
Individual IV-2, who was markedly affected, had a severe and widespread muscle
atrophy with fat replacement in the legs (most severe in gastrocnemius and soleus), a
moderate degree of muscle involvement in the thighs (mainly vasti, biceps femoris,
and semitendinosus muscles), and a mild fatty infiltration of gluteal maximus muscle
(Figure 2C).
To explore the influence of the p.Gly269Val mutation on the TFG function, we
conducted in vitro transfection analysis. We subcloned a 1.2 kb PCR-amplified
fragment containing the coding region of TFG from human cDNA libraries into
pFLAG-CMV-5a (Sigma-Aldrich) to construct TFG expression plasmids (Table S2).
The mutation c.806G>T (p.Gly269Val) was introduced by using QuikChange
Site-Directed Mutagenesis method (Stratagene) (Table S2). To investigate the
expression of both wild-type and mutant TFG proteins, we incubated human
embryonic kidney (HEK) 293 cells maintained in Dulbecco’s modified Eagle’s
medium (DMEM; Hyclone) containing 10% fetal bovine serum (FBS; Invitrogen) at
37oC and in 5% CO2 and transfected them with plasmids expressing wild-type or
Gly269Val TFG by using calcium-phosphate precipitation. At 48 hours after
transfection, total-protein cell extracts were analyzed by Western blotting analysis,
which showed that the steady-state level of the Gly269Val mutant TFG was
significantly lower than that of wild-type TFG (Figure 3A, 3B; Table S3).
Since reduced steady-state level of mutant TFG proteins may result from an
enhanced protein degradation, a reduced mRNA synthesis, or formation of detergent
9
insoluble protein aggregates19, we next evaluated the impact of the p.Gly269Val
mutation on TFG degradation by a cycloheximide-chase assay as previously
described,6 and found that the mutation did not compromise the protein stability
(Figure S2). Further analysis of TFG mRNA levels in HEK293 cells expressing
wild-type or Gly269Val TFG by real-time quantitative PCR analysis (RT-qPCR)
revealed no significant difference between the wild-type and the mutant TFG mRNA
levels (Figure 3C; Table S2). The TFG mRNA levels of the dermal fibroblasts from
the affected individual IV-2 and an age-matched male healthy control subject were
also similar (Figure S3), indicating that the p.Gly269Val mutation does not affect the
quantity of TFG mRNA. Then, we utilized Aggrescan, a bioinformatic tool, to
evaluate whether the mutation may increase the propensity of TFG proteins to form
aggregates.20 In silico analysis predicted that the TFG p.Gly269Val mutation might
increase the aggregation tendency of TFG by generating a new aggregation-prone
region at the residues 269 to 274.
To elucidate whether the reduced levels of Gly269Val TFG in the transfected
cells is resulting from the enhanced tendency of the TFG mutant proteins to form
aggregates, we examined the solubility of wild-type TFG and TFG mutants in
HEK293 cells. We also used Pro285Leu TFG as a control because previous study
showed that its expression level in HEK293T cells was not different from that of
wild-type TFG.18 The cells transfected with different TFG constructs were extracted
with RIPA buffer (soluble fraction) first and subsequently extracted with urea buffer
(7M urea, 2M thiourea, 1% ASB-14, 40mM Tris, pH 8.5) to recover insoluble TFG.
Western blotting analysis revealed that only the Gly269Val TFG was predominantly
found in the insoluble fraction of cell lysates, whereas the wild-type protein and the
Pro285Leu mutant were detected predominantly in the soluble fraction (Figure 4A
10
and 4B). Similar results were obtained when experiments were performed using HeLa
cells (Figure 4A and 4B), indicating that the Gly269Val TFG had such a strong
tendency to form detergent-insoluble aggregates that its soluble form in cytosol was
significantly depleted, while the Pro285Leu TFG as well as wild-type TFG did not
have such propensities. The differences in biochemical characteristics between
Gly269Val TFG and Pro285Leu TFG may partially explain why they are associated
with disparate clinical phenotypes (CMT2 and HMSN-P).
To clarify the intracellular features of both wild-type and Gly269Val TFG, we
performed immunofluorescence analyses of HEK293 cells transfected with wild-type
or the mutant TFG constructs (Table S3). Cell expressing wild-type TFG displayed
numerous punctate TFG-specific staining throughout the cytosol, whereas cells
expressing the Gly269Val TFG demonstrated multiple large cytoplasmic aggregates
with TFG-specific staining (Figure 3D). These cytoplasmic aggregates were negative
for TDP-43 staining (data not shown). Both wild-type TFG and Gly269Val TFG had a
similar subcellular localization and were mainly expressed in the ER, evidenced by
both colocalizing with DesRed-ER (Clontech), an ER marker (Figure S4A), and
Sec16B, a marker of ER exit sites (Figure S4B).16 Since wild-type TFG molecules
form oligomers in their normal functioning, we investigated the association between
wild-type TFG and Gly269Val TFG proteins. Equal amounts of plasmids encoding
DesRed-tagged wild-type TFG and GFP-tagged Gly269Val TFG were co-transfected
into HEK293 cells. Co-localization of wild-type and mutant TFG within cytoplasmic
aggregates suggests that the Gly269Val TFG molecules did not lose their ability to
congregate with wild-type TFG in an oligomeric complex (Figure S4C).
Since the fluorescent analysis revealed that wild-type TFG and the Gly269Val
mutant co-localized within the cytoplasmic aggregates in the cells (Figure S4C), the
11
mutant TFG may incorporate the wild-type protein to form aggregates. To evaluate
the impact of such effect on cells harboring the heterozygous p.Gly269Val TFG
mutation, we compared the level of soluble cytoplasmic TFG of HEK293 cells
transfected with wild-type TFG construct and that of cells transfected with half dose
of the wild-type construct and half dose of the mutant construct. Western blotting
analysis revealed that the TFG level of the cells expressing both wild-type and mutant
TFG was only approximately one third of that of the cells expressing solely wild-type
TFG (Figure S5A and B), indicating that the Gly269Val mutant may partially deplete
wild-type TFG through the aggregate formation process.
In addition, accumulated cytoplasmic protein aggregates may provoke ER stress
or lead to cellular toxicity. Thus, we examined whether the Gly269Val TFG mutants
affected cell viability. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) colorimetric assay was used to assess cell viability of HEK293 cells
transfected with vectors expressing wild-type TFG or Gly269Val TFG, or empty
vector. We found that cells expressing Gly269Val TFG did not have significant
reduction in viability when compared with the cells expressing wild-type TFG or the
cells transfected with empty vector (Figure S6A), suggesting that Gly269Val TFG did
not have a significant cellular toxicity.
To investigate whether the Gly269Val mutant may induce ER stress or activate
the unfolded protein response (UPR), biomarkers of ER stress, such as expression of
ER resident chaperon Bing Ig protein (BiP)21 and transcriptional factor CAATT
enhancer-binding protein homologous protein (CHOP)22 and alternative splicing of
X-box binding protein I (XBP-1),23 were examined in the transfected HEK293 cells
(Table S2). Cells exposed to a stress stimulator dithiothreitol (DTT) or transfected to
express Arg89Cys myelin protein zero, a previously identified ER stress-inducing
12
mutant protein,24 were used as the positive controls. As shown in Figure S6B, cells
expressing Gly269Val TFG did not have a significantly higher level of Bip or spliced
XBP-1 compared with the cells expressing wild-type TFG, suggesting that the
Gly269Val TFG did not induce ER stress, or increase the cellular susceptibility to ER
stress. Under treatment of 1 mM DTT, the levels of BiP, CHOP, or spliced XBP-1 did
not increase in the cells expressing Gly269TFG compared with those expressing
wild-type TFG (Figure S6C).
Because Gly269Val TFG may partially deplete wild-type TFG and its
overexpression did not increase cellular toxicity, the pathogenesis of the heterozygous
TFG p.Gly269Val mutation may come from low soluble level of cytoplasmic TFG,
which may result in insufficient functional TFG molecules. To test the impact of
depletion of wild-type TFG on cells, we designed siRNA specifically targeting the
3’UTR of TFG to efficiently suppress the expression of endogenous wild-type TFG
(Figure S7, Table S2). Utilizing MTT assay, we examined the cell viability of the
HEK293 cells co-transfected with the siRNA and plasmids expressing wild-type TFG
or Gly269Val TFG, or empty vectors, and found that depletion of endogenous TFG
significantly reduced in vitro viability, which could be rescued by expressing
exogenous wild-type TFG but not Gly269Val TFG (Figure S8).
Previous studies have shown that TFG depletion slows protein secretion from the
ER and alters the ER structure.16,17 To investigate the effect of the Gly269Val mutant
on the protein secretory pathway, we co-transfected HEK293 cells with reporter
plasmids encoding Gaussia luciferase (Gluc) and Firefly luciferase (Fluc), along with
plasmids expressing wild-type or Gly269Val TFG or empty vector. The Gaussia
luciferase can be secreted out of the cells and has been commonly utilized to monitor
protein secretory pathway25. We measured the Gluc activities in the culture medium
13
of the cells at 48 hours after transfection with wild-type, Gly269Val TFG, or empty
constructs, and normalized the Gluc activities with the corresponding internal Fluc
control activities. The luciferase assay revealed that there was no significant
difference between the secretory efficiencies of Gluc between the cells expressing
wild-type TFG, mutant TFG, or empty vector (Figure 5A). Since the endogenous TFG
of the HEK293 cells might have been sufficient for the protein secretory function, the
siRNA targeting the 3’UTR of TFG was used to inhibit endogenous TFG of cells
transfected with different TFG constructs or empty vectors and the secretion
efficiencies of Gluc in cells were investigated. The results indicated that inhibition of
endogenous TFG compromised the protein secretory pathway, which could be rescued
by replenishing with exogenous wild-type TFG but not Gly269Val TFG (Figure 5B).
These findings support that cells with a heterozygous TFG p.Gly269Val mutation may
have compromised protein secretory function, which may lead to a decrease in
cellular viability.
Utilizing exome sequencing, bioinformatics filtering and co-segreation analysis,
we have identified only one heterozygous mutation, TFG p.Gly269Val, in this large
pedigree with autosomal dominant CMT2. This mutation alters the conserved amino
acid residue, is absent in the 6,500 exomes from the NHLBI Exome Variant Server,
and does not occur in 1,140 ethnic control chromosomes. In vitro transfection studies
revealed that Gly269Val TFG mutant proteins tended to form large intracellular
aggregates and depleted functional TFG molecules by sequestrating wild-type TFG
into aggregates. Depletion of TFG in cells by siRNA compromised the protein
secretory function and decreased cell viability, which both could be rescued by
expression of exogenous wild-type TFG but not the Gly269Val mutant. This study
provides strong genetic and functional evidences to support that the TFG p.Gly269Val
14
mutation causes CMT2 and thus, identifies TFG as a new member on the list of
CMT2-associated genes.
TFG is ubiquitously expressed in human neuronal and non-neuronal cells, as
well as in the spinal motor neurons and dorsal root ganglia.15 Only two mutations in
TFG have been previously identified. One is the heterozygous p.Pro285Leu mutation
which causes HMSN-P and another is the homozygous p.Arg106Cys mutation which
results in a complicated form of recessive HSP, presenting as spastic paraplegia, optic
atrophy, and neuropathy (SPOAN).15,17 Both mutations are associated with
neurological diseases, indicating that TFG is critically important for neuronal function.
Our work provides additional genetic evidence that the heterozygous p.Gly269Val
mutation in TFG could cause a dominant axonal CMT. Thus far, more than 60% of
the genetic causes of axonal CMT remain unknown, reflecting the molecular
complexity of the structure and function of peripheral axons and neurons. Although
the full scopes of TFG functions are still unclear, recent studies revealed that TFG
functions at ER exit sites (ERES) and regulates secretory protein vesicle biogenesis
and egress from the ER.16 In neuronal cells, properly functioning ERES helps provide
sufficient protein cargoes for the axonal transport system and enable efficient
secretion of locally synthesized proteins from the axonal ER.26 ERES also plays an
important role in supporting axonal outgrowth.27 Therefore, TFG dysfunction may
compromise protein secretory pathway and result in a shortage or insufficiency of
local functional proteins in axons, which may be the common consequence of a host
of hereditary axonopathies associated with mutations in the genes involved in axonal
transport (such as HSPB1, NEFL, BICD2, DYNC1H1, KIF5A, KIF1A, MYH14), or
ER and Golgi organization (such as BSCL2, REEP1, ATL1, FAM134B).3,28
It is intriguing that three different mutations in TFG cause three disparate clinical
15
phenotypes, implying that each mutation has its unique pathogenic mechanisms. The
TFG p.Gly269Val mutation, which is associated with a dominant CMT2, increases the
aggregation propensity of TFG proteins. In cells harboring the heterozygous
p.Gly269Val mutation, mutant TFG proteins tend to form cytoplasmic aggregates,
which also sequestrate a sizable wild-type TFG proteins and lead to insufficient
soluble TFG molecules available to maintain their proper functions. The TFG
p.Gly269Val mutation seems not to have a toxic gain-of-function, demonstrated by
that in vitro overexpression of the mutant TFG did not affect cell viability. However,
its dominant-negative effect in neuronal cells with the heterozygous mutation may
deplete wild-type TFG molecules and compromise protein secretory process, with the
neurons with the longest axons most vulnerable and leading to the distal symmetric
motor and sensory symptoms in the CMT2. The TFG p.Arg106Cys mutation, which
is associated with a recessive complicated HSP presenting as SPOAN, abolishes the
ability of TFG proteins to self-assemble into a functional oligomer.17 Individuals
carrying heterozygous TFG p.Arg106Cys mutation are asymptomatic, suggesting that
one copy of wild-type TFG is sufficient to produce enough proteins for physiological
function. Compared with those harboring the heterozygous TFG p.Gly269Val
mutation, the individuals carrying the homozygous TFG p.Arg106Cys mutation may
have a much more profound loss of TFG function, which thus causes a widespread
axonopathy involving corticospinal tracts, peripheral nerves and optic nerves. In
addition, the heterozygous TFG p.Pro285Leu mutation involved in dominant
HMSN-P may pose a toxic gain-of-function effect through a possible RNA-mediated
and TDP-43 related mechanism.15 The Pro285Leu TFG protein may elicit a direct
toxic effect on neuronal cells to cause the motor and sensory symptoms. The
explanation for selective proximal involvement in HSMN-P remains elusive.
16
In conclusion, our findings demonstrate a novel TFG mutation causing
autosomal dominant CMT2 and highlight the importance of TFG in protein secretory
pathways which are essential for proper functioning of human peripheral nervous
system.
Supplemental Data
Supplemental Data, including eight figures and three tables, can be found with this
article online at http://www.cell.com/AJHG/.
Acknowledgments
The authors thank the patients who participated in this study. This work was
supported by research grants from the National Science Council, Taiwan, ROC
(NSC102-2628-B-075-006-MY3), the Ministry of Education, Aim for the Top
University Plan (V100-E6-006, V100-E6-007 and V101E7-005), the Brain Research
Center, National Yang-Ming University, the High-throughput Genome Analysis Core
Facility and Bioinformatics Consortium of Taiwan of National Core Facility Program
for
Biotechnology,
Taiwan
(NSC100-2319-B-010-001
NSC100-2319-B-010-002).
Web Resources
The URLs for data presented herein are as follows.
1000 Genomes Project, http://www.1000genomes.org
Aggrescan, http://bioinf.uab.es/aap/
dbSNP, Human Build 132, http://www.ncbi.nlm.nih.gov/projects/SNP/
GeneNote, http://bioinfo2.weizmann.ac.il/cgi-bin/genenote/ home_page.pl
and
17
GRCh37 patch 2, obtained from ftp://ftp.ensembl.org/pub/release-61/
NHLBI Exome Sequencing Project, http://evs.gs.washington. edu/EVS/
Online Mendelian Inheritance in Man (OMIM), http://www.omim.org/
Panther, http://www.pantherdb.org/tools/csnpScoreForm.jsp/
PMut, http://mmb.pcb.ub.es/PMut/
SNAP, https://www.rostlab.org/services/snap/
SNPs&GO, http://snps-and-go.biocomp.unibo.it/snps-and-go/
UniGene, http://www.ncbi.nlm. nih.gov/unigene/
18
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