A novel homozygous MPV17 mutation in two families with axonal sensorimotor polyneuropathy
Choi et al. BMC Neurology
A novel homozygous MPV17 mutation in two families with axonal sensorimotor polyneuropathy
Yu-Ri Choi 3
Young Bin Hong 2
Sung-Chul Jung 3
Ja Hyun Lee 0
Ye Jin Kim 0
Hyung Jun Park 6
Jinho Lee 1
Heasoo Koo 5
Ji-Su Lee 1
Dong Hwan Jwa 1
Namhee Jung 3
So-Youn Woo 4
Sang-Beom Kim 8
Ki Wha Chung 0
Byung-Ok Choi 1 7
0 Department of Biological Science, Kongju National University , 56 Gonjudaehak-ro, Gongju, Chungnam 314-701 , Korea
1 Department of Neurology, Samsung Medical Center, Sungkyunkwan University School of Medicine , 81 Irwon-ro, Gangnam-Gu, Seoul 135-710 , Korea
2 Stem Cell & Regenerative Medicine Center, Samsung Medical Center , Seoul , Korea
3 Department of Biochemistry, Ewha Womans University School of Medicine , Seoul , Korea
4 Department of Microbiology, Ewha Womans University School of Medicine , Seoul , Korea
5 Department of Pathology, Ewha Womans University School of Medicine , Seoul , Korea
6 Department of Neurology, Ewha Womans University School of Medicine , Seoul , Korea
7 Neuroscience center, Samsung Medical Center , Seoul , Korea
8 Department of Neurology, Kyung Hee University, College of Medicine , Seoul , Korea
Background: Mutations in MPV17 cause the autosomal recessive disorder mitochondrial DNA depletion syndrome 6 (MTDPS6), also called Navajo neurohepatopathy (NNH). Clinical features of MTDPS6 is infantile onset of progressive liver failure with seldom development of progressive neurologic involvement. Methods: Whole exome sequencing (WES) was performed to isolate the causative gene of two unrelated neuropathy patients (9 and 13 years of age) with onset of the syndrome. Clinical assessments and biochemical analysis were performed. Results: A novel homozygous mutation (p.R41Q) in MPV17 was found by WES in both patients. Both showed axonal sensorimotor polyneuropathy without liver and brain involvement, which is neurophysiologically similar to axonal Charcot-Marie-Tooth disease (CMT). A distal sural nerve biopsy showed an almost complete loss of the large and medium-sized myelinated fibers compatible with axonal neuropathy. An in vitro assay using mouse motor neuronal cells demonstrated that the abrogation of MPV17 significantly affected cell integrity. In addition, the expression of the mutant protein affected cell proliferation. These results imply that both the loss of normal function of MPV17 and the gain of detrimental effects of the mutant protein might affect neuronal function. Conclusion: We report a novel homozygous mutation in MPV17 from two unrelated patients harboring axonal sensorimotor polyneuropathy without hepatoencephalopathy. This report expands the clinical spectrum of diseases caused by mutations of MPV17, and we recommend MPV17 gene screening for axonal peripheral neuropathies.
Mitochondrial DNA depletion syndrome 6 (MTDPS6); MPV17; Navajo neurohepatopathy (NNH); Sensorimotor polyneuropathies; Whole exome sequencing (WES)
Mutations in MPV17 cause mitochondrial DNA depletion
syndrome 6 (MTDPS6) (NNH; MIM #256810), also known
as Navajo neurohepatopathy, an autosomal, recessive,
multi-system disorder . MTDPS6 is divided into three
clinical phenotypes based on age at onset and the course of
the disease: infantile form (onset before age of 6 months)
with jaundice and failure to thrive before 2 years of age,
childhood form (onset between age 1 and 5 years) resulting
in early death from liver failure, and the classical form with
moderate liver dysfunction and progressive neuropathy .
The function of MPV17 protein remains uncertain.
Further investigation is needed to determine whether the
disease is primarily a neurologic disorder or a consequence of
a metabolic disorder. MPV17 encodes a mitochondrial,
inner membrane protein, and it has been implicated in the
metabolism of oxidative phosphorylation (OXPHOS),
glycogen storage, mitochondrial morphology, and the integrity of
mitochondrial DNA (mtDNA) under stress conditions .
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An MPV17-deficient mouse model exhibited severe
mtDNA depletion in skeletal muscle and the liver, but not
in the brain or kidneys. Other phenotypes of NNH, such as
liver cirrhosis or failure, are the most common, but have
not occurred. However, the mice exhibited sensorineural
deafness due to severe degeneration of the inner ear, and
with associated apoptosis of the outer hair cells [4–6]. These
results imply the significance of MPV17 in cellular integrity.
In addition, in the absence of MPV17, yeast cells became
more vulnerable to metabolic or oxidative stresses .
Although the incidence of the neurohepatopathy by
MPV17 mutations in the western Navajo Reservation is
relatively frequent (1 in 1,600 live birth), a few non-Navajo
patients have been investigated [8, 9]. The presentation of
NNH revealed multi-systemic disorder, and the patients
with classical form showed axonal sensorimotor
neuropathies with other clinical manifestations, such as liver
disease, diabetic mellitus, or brain abnormalities.
Here, we report on two unrelated patients with MPV17
homozygous mutation. In contrast to previously reported
NNH patients [2, 8–11], the patients presented here
exhibited only axonal sensorimotor polyneuropathy without liver
and brain symptoms. This is the first report of MPV17
dysfunction in Korean patients, and we analyzed the
detrimental effect of mutant MPV17 on a motor neuronal cell line.
This study enrolled two autosomal recessive Korean
families with axonal sensorimotor polyneuropathy (FC26
and FC355, Fig. 1a). After careful clinical and
electrophysiological examinations, 300 healthy controls were
recruited from the neurological department. All
participants provided written, informed consents according to
the protocol approved by the Institutional Review Board
for Ewha Womans University, Mokdong Hospital (ECT
11-58-37). In addition, the patients provided written,
informed consent for the publication of individual
clinical details, and for the publication of family trees.
Fig. 1 Pedigree, sequencing chromatograms, and conservation analysis. a Pedigrees of FC26 (left) and FC355 families (right). Genotypes of MPV17
c.122G> A mutation were indicated bottom of each examined individuals. The open symbols represent unaffected individuals and filled symbols
represent affected individuals. Asterisks indicate samples whose DNA were used for WES. b Confirmation of the mutation by capillary sequencing
method. Vertical arrows indicate the mutation site. c Conservation analysis of mutation site in MPV17. The mutation site (R41, yellow) and adjacent
amino acid sequences are well conserved across species. R50 and transmembrane domains are indicated in green and gray colors, respectively
(H. sapiens: NP_002428.1, M. musculus: NP_032648.1, R. norvegicus: NP_001091710.1, B. taurus: NP_001039394.1, G. gallus: XP_004935875.1, P. bivittatus:
XP_007420911.1, X. laevis: AAH82223.1, and D. rerio: NP_957459.2)
Two independent neurologists evaluated each patient,
and collected clinical information including assessments
of motor and sensory impairments, deep tendon
reflexes, and muscle atrophy. Muscle strength of flexor
and extensor muscles were assessed manually using the
standard medical research council (MRC) scale. In order
to detect any physical disability we used a nine-point
functional disability scale (FDS) , which was based
on the following criteria: 0: normal; 1: normal but with
cramps and fatigability; 2: an inability to run; 3: walking
difficulty but still possible unaided; 4: walking with a
cane; 5: walking with crutches; 6: walking with a walker;
7: wheelchair bound; and 8: bedridden. Sensory
impairments were assessed for the level and severity of pain,
temperature, vibration and position. Age at onset was
determined by asking patients for their ages, when
symptoms first appeared.
Motor and sensory conduction velocities of median,
ulnar, peroneal, tibial, and sural nerves were determined.
Motor conduction velocities (MCVs) of the median and
ulnar nerves were determined by stimulating at the
elbow and wrist, while recording compound muscle
action potentials (CMAPs) over the abductor pollicis
brevis and adductor digiti quinti, respectively. In the same
way, the MCVs of peroneal and tibial nerves were
determined by stimulating at the knee and ankle, while
recording CMAPs over the extensor digitorum brevis and
adductor hallucis, respectively. Sensory conduction
velocities (SCVs) were obtained over a finger-wrist segment
from the median and ulnar nerves by orthodromic
scoring, and were also recorded for sural nerves. Sensory
nerve action potential (SNAP) amplitudes were
measured from positive peaks to negative peaks.
Sural nerve biopsy
Distal sural nerve was biopsied from patient 1 (Fig. 1a,
II-1) at 34 years, and pathological examination included
light and electron microscopic analyses. Formalin-fixed
sections were stained with hematoxylin and eosin
(H&E), modified Masson’s trichrome, and Luxol fast
blue. For electron microscopic study, the specimen was
fixed in 2 % glutaraldehyde in 25 mM cacodylate buffer.
Semithin sections were stained with toluidine blue and
ultra-thin cut samples were contrasted with uranyl
acetate and lead citrate.
Vastus lateralis muscle biopsy
Cross-sections of the biopsy of the vastus lateralis
muscle from patient two (Fig. 1b, III-2) at 22 years
were stained with H&E, modified Gomori-trichrome,
NADH-tetrazolium reductase (NADH-TR), succinate
dehydrogenase (SDH), periodic acid Schiff (PAS),
Oilred-O and adaenosine triphosphatase reaction, and
immunostained for myosin heavy chain (Vision
Biosystems, Newcastle, UK). Samples were also examined
using electron microscopy.
MR images of brain, hip, thigh and lower leg
Brain, hip, thigh and lower leg of both patients were
evaluated using a 1.5-T system (Siemens Vision; Siemens,
Erlangen, Germany). Whole brains were scanned using a
slice thickness of 7 mm and 2-mm interslice gap, to
produce 16 axial images. The imaging protocol consisted of
T2-weighted spin echo (SE) (TR/TE = 4,700/120 ms),
T1-weighted SE (TR/TE = 550/12 ms), and
fluidattenuated inversion recovery (FLAIR) (TR/TE = 9,000/
119 ms, inversion time 2,609 ms) images. Images of the
hip, thigh and lower leg were obtained in axial [field of
view (FOV) 24–32 cm, slice thickness 6 mm, and slice
gap 0.5–1.0 mm] and coronal planes (FOV 38–40 cm,
slice thickness 4–5 mm, slice gap 0.5–1.0 mm). xThe
following protocol was used: T1-weighted SE (TR/TE
570–650/14–20, 512 matrices), T2-weighted SE (TR/TE
2800–4000/96–99, 512 matrices), and fat-suppressed
T2weighted SE (TR/TE 3090–4900/85–99, 512 matrices).
Exome sequencing and filtering
Whole exome sequencing (WES) was performed for six
samples (three from each family), according to a
previous study . Briefly, WES was performed using the
Human SeqCap EZ Human Exome Library v3.0 (Roche/
NimbleGen, Madison, WI, USA), and the HiSeq 2000
Genome Analyzer (Illumina, San Diego, CA, USA). The
UCSC assembly hg19 was used as the reference
sequence and variant calling was achieved in cases with
>20 single nucleotide polymorphisms (SNP). We
collected functionally significant variants (missense,
nonsense, exonic indel and splicing site variants) from about
70 peripheral neuropathy genes and 15 mitochondrial
DNA depletion syndrome (MTDPS)-related genes, and
then variants agreeing with autosomal recessive
inheritance were selected. Causative variants were confirmed
by the Sanger’s sequencing method, and conservation
analysis of mutation sites was performed using the
MEGA5 program, ver 5.05
(http://www.megasoftware.net/). In silico analyses were performed using the
prediction algorithms SIFT (http://sift.jcvi.org) and MUpro
Construction of wild-type and mutant MPV17
To obtain the MPV17 transcript, cDNA was synthesized
using Superscript reverse transcriptase (Invitrogen,
Carlsbad, CA, USA) from total mRNA of HEK293. Then
polymerase chain reaction (PCR) was performed using
the cDNA as a template. The amplified PCR product
was cloned into the expression vector, pCMV-myc
(Clontech, Mountain View, CA, USA). Mutant MPV17
transcript were generated by QuikChange Site-Directed
Mutagenesis Kit (Stratagene, La Jolla, CA, USA). All
primers’ sequences are listed in Additional file 1: Table S1.
Transfection and knockdown of MPV17
NSC34 cells were cultured in a 10 % FBS, 1 % PS and high
glucose Dulbecco’s modified eagle medium (DMEM;
Biowest, Nuaille, France). To express MPV17 transcript in
the motor neuron, NSC34 cells were transfected with
MPV17 DNA-containing vectors using Lipofectamine
2000 reagent (Invitrogen), according to the manufacturer’s
recommendation. Knockdown of MPV17 was performed
using MPV17-specific siRNA and Lipofectamine 2000
reagent (Invitrogen) (Additional file 1: Table S1). Cells were
harvested after overexpression and knockdown of MPV17
at 24 and 72 h.
Measurement of proliferation and cell viability
After 3 days of knockdown, NSC34 cells were transferred
to 24-well plates. Then, the proliferation of the cells was
determined by direct counting under a microscope at 24 h
intervals. For the overexpression model, NSC34 cells,
cultured on 24-well plates, were transfected with wild-type or
mutant MPV17. Cells were counted at 24 h intervals.
Sensitivity to H2O2 was measured by a
3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly,
cells treated with H2O2 were incubated with 10 mM MTT
solution for 2 h, then the cells were lysed with dimethyl
sulfoxide. Relative numbers of viable cells were determined
using absorbance at 560 nm.
Protein synthesis in NSC34 cells was determined using
standard Western blotting with anti-myc Ab (Abcam,
Cambridge, UK), anti-actin Ab, anti-mouse secondary
Ab, and anti-rabbit secondary Ab (Sigma, St. Louis, MO,
USA). An OXPHOS detection cocktail (Abcam) was
used based on standard Western blotting. ECL plus
Western blotting substrate (Thermo Scientific, Rockford,
IL, USA) were used for detection of proteins.
Identification of a novel homozygous mutation in MPV17
The mean sequencing yields of six WES data was
approximately 11.18 Gb/sample with mappable reads of 96 %.
Approximately, 89,235 variants (SNPs and indels) were
observed from each sample. Of these, 20,775 were located
in a coding region (Additional file 1: Table S2).
After filtering the WES data for 3 members in each
family (Fig. 1), a novel homozygous mutation c.122G> A
(p.R41Q) in MPV17 was identified in both families (Fig. 1b).
A mutant allele was putatively inherited from each parent
in both families. The mutation was not observed in the 300
controls and in-house exome data (n = 302). In addition, it
was not registered in the dbSNP142
(http://www.ncbi.nlm.nih.gov) or the 1000 Genomes Project Database (http://
www.1000genomes.org/). However, this mutation was
reported in the Exome Sequencing Project database (http://
evs.gs.washington.edu/EVS/) and ExAc browser (http://
exac.broadinstitute.org/) with very low allele frequency
(0.00008 and 0.00002471, respectively). The mutation site
was well-conserved across vertebrate species (Fig. 1c). In
silico analysis of the mutation was predicted to affect the
protein stability by SIFT (0.02) or MUPro (−0.422)
programs. Although more than 40 functionally significant
variants were found in ~70 CMT- and ~15 MTDPS-related
genes, they were not considered the genetic cause, except
for the MPV17 mutation, because they were found in the
controls or noncosegregated with affected individuals
(Additional file 1: Table S3 and S4).
A 34-year-old woman (FC26; Fig. 1a, II-1) was the first
child of healthy non-consanguineous Korean parents.
The proband was born at full term and the perinatal
histories were unremarkable. Early motor milestones were
not delayed, and 1 year after her birth, she was able to
walk. At 9 years of age, she first noticed muscle
weakness of the distal lower limbs. She began to walk with
short leg braces at 12 years of age. Neurologic
examination at the age of 34 years revealed muscle weakness
and atrophies of bilateral, distal muscles, predominantly
in the lower limbs. Bilateral, severe atrophic changes of
the intrinsic hand, foot and calf muscles, and flexion
deformities of interphalangeal joints were noted (Fig. 2a-c).
Ankle joint deformity and scoliosis was observed
although she was able to walk with orthopedic assistance.
Vibration and position senses were more severely
disturbed than pain and touch senses. Knee and ankle jerks
were absent. No pyramidal or cerebellar signs were
detected. However, she did not present any other clinical
presentations of NNH, such as growth retardation,
gastrointestinal dysmotility, hepatomegaly, cognitive
impairment, ophthalmoplegia, corneal scarring or hypoglycemic
attacks. Elevated serum lactate levels were revealed
(2.1 mmol/L, reference value: < 1.6 mmol/L), but the
serum levels of liver enzyme, glucose and pyruvate were
A 22-year-old man (FC355; Fig. 1b, III-2) was the second
child of healthy non-consanguineous parents. Early
motor milestones were normal, as in patient one. At age
13, he first felt gait and balance problems. He began to
Fig. 2 Hand and leg pictures of patients. a, b Hands of patient 1 (a) and 2 (b). Right hand showed severe, atrophied, intrinsic muscle and flexion
deformities of interphalangeal joints. c, d Lower extremities of patient 1 (c) and 2 (d). Severe bilateral muscle atrophies and weakness with ankle
joint deformities were observed in both patients
walk with short leg braces at 20 years of age. Neurologic
examination at the age of 22 years revealed muscle
weakness and atrophies of the bilateral distal muscles.
Bilateral flat feet, and atrophic changes of intrinsic hand,
foot and calf muscles were also noted (Fig. 2d). Like
patient one, he complained that vibration and position
senses were more severely disturbed than pain and
touch senses, and he was able to walk with assistance.
Deep tendon reflexes, in all extremities, were absent. He
did not present with other phenotypes of NNH.
Laboratory findings were normal except for a slightly elevated
serum lactate (1.9 mmol/L).
Sensory nerve involvement in the early stage of disease
An electrophysiological study showed similar results in
both patients (Table 1). Sensory-nerve action potentials
of the sural nerve were lost in the early stage of the
disease. MCVs and CMAPs were decreased in median and
ulnar nerves and CMAPs of peroneal and tibial nerves
were not elicited. SCVs and SNAPs were not elicited in
either patient. Visual evoked potential (VEP) and
brainstem auditory evoked potential (BAEP) were normal.
Fatty replacement in the soleus muscle
MR imagery at the thigh level of patient one showed
that the vastus lateralis muscle was mildly affected
compared to other muscles (Fig. 3a, arrow), while nearly all
muscles were normal in patient two (Fig. 3b). Lower calf
muscle MRIs showed predominant and severe muscle
atrophies and fatty replacements in the soleus muscles
of both patients; however, the tibialis posterior and
lateral gastrocnemius muscles were relatively sparing
(Fig. 3c and d). It is noteworthy that both patients
showed similar MRI patterns: T1-weighted images showed
marked fatty infiltration in the lower calf muscles
compared to the thigh and the hip, which was consistent with
the length-dependent axonal degeneration. Brain MRI did
not reveal any abnormality in either patient.
Marked loss of large and medium sized myelinated fibers
Semithin transverse sections from the left distal sural
nerve biopsy, from patient 1, showed several remaining
small myelinated fibers (MFs) with complete loss (10/
mm ) of large and medium-sized MFs (normal distal
sural nerve in 32-year-old female: 9,200/mm2) (Fig. 4a).
Electron microscopic examination showed atrophy and
occasional vesicular changes in the unmyelinated axons.
There was no evidence of demyelination, remyelination
or onion bulb formation (Fig. 4b).
Chronic myopathy with few ragged red fibers
NADH-TR (data not shown) and SDH (Fig. 4c) stain in
the left vastus lateralis muscle biopsy of patient two,
showed increased irregular positive reaction, and
modified Gomori trichrome stain (data not shown) showed a
few ragged, red fibers. Electron microscopic examination
showed myofibers with focal subsarcolemmal
accumulation of enlarged mitochondria and abnormal
membranous structure (Fig. 4d).
Mutant protein inhibits cell proliferation and viability
To investigate the role of MPV17 in motor neurons, we
measured the effect on cell proliferation after abrogation
of MPV17. Transfection of MPV17-specific siRNA for
72 h efficiently reduced the mRNA level in NSC34, a
mouse motor neuronal cell line (Fig. 5a). In this setting,
knockdown of MPV17 significantly reduced the cell
proliferation (Fig. 5b) and viability against ROS in mouse
Table 1 Electrophysiological features of the patients with MPV17 mutation
Age at exam (years)
Median sensory nerve
Ulnar sensory nerve
Abbreviations: A absent potentials, TL terminal latency, CMAP compound muscle action potential, MNCV motor nerve conduction velocity, SNAP sensory nerve
action potential, SNCV, sensory nerve conduction velocity, ND not done. Bold character indicates abnormal values
motor neuronal cells (Fig. 5c). Abrogation of MPV17 also
affected mitochondrial integrity, which was observed by
amounts of OXPHOS (Additional file 2: Figure S1a). Next,
in order to investigate the effect of MPV17 mutations,
we cloned wild-type MPV17 gene and generated
mutations including p.R41Q. Expression of the proteins in
NSC34 was confirmed by Western blotting (Fig. 5d).
Expression of p.R41Q mutant protein significantly
inhibited cell proliferation when compared to controls
(Fig. 5e). In addition, expression of p.R50Q and p.L143*
proteins significantly reduced cell proliferation, whereas
p.KM88-89ML mutant exhibited a mild effect.
Overexpression of the mutants mildly affected mitochondrial
OXPHOS system in the presence of endogenous
MPV17 protein (Fig. 5f ).
Clinical features of the present patients are considerably
different from those of previously described NNH
patients with MPV17 mutations. The disease presentation
has frequently been described as axonal sensorimotor
neuropathies; however, the neuropathies were always
associated with hepatopathy, or brain involvements, in the
classical form [14-15]. But, the present two patients
showed only axonal sensorimotor polyneuropathy, and
they did not exhibit any liver or brain abnormalities, at
the ages of 34 and 22 years. However, these patients are
young and other organ systems may eventually become
involved, so follow-up monitoring is needed. We also
observed severe impairment of the hands and lower legs
with severe muscle atrophy and contractures due to
Fig. 3 Lower extremity MRI of patients. a, b T1-weighted axial MRIs of the thigh and (c, d) lower leg of patient 1 (a and c) and 2 (b and d). At
the thigh level, muscle atrophies and hyperintense signal changes were shown in the vastus lateralis muscle (arrow). However, the lower leg MRIs
revealed diffuse fatty hyperintense signal changes in the soleus muscles (arrowhead), but the tibialis posterior and gastrocnemius muscles were
peripheral neuropathy. In addition, lower extremity MRI
revealed severe distal fatty infiltration, which is consistent
with length-dependent axonal degeneration. Moreover, the
lower leg MRIs exhibit selective fatty infiltration with a
preference for soleus muscles, similar to CMT type 2A
(CMT2A) caused by MFN2 mutations .
Currently, there is limited information on the function of
MPV17. The MPV17−/− mouse model exhibited peripheral
neuropathy, with sensorineural deafness and kidney failure
[6, 17], which is in good agreement with the human
phenotype. In addition, the loss of MPV17 resulted in apoptosis
in outer hair cells, which implies the significance of MPV17
in cellular viability [4–6]. To address the function of
MPV17 in the peripheral nervous system (PNS), we first
confirmed the expression of MPV17 in human PNS using
transcriptome data from human sural nerve (data not
Fig. 4 Histopathological characterization of sural nerve and vastus lateralis muscle. Sural nerve (a and b) from patient 1, and vastus lateralis muscle
(c and d) biopsies from patient two were performed at 34 and 22 years, respectively. a Semi-thin transverse section. Toluidine blue stain shows the absence
of large and medium myelinated fibers with rarely-noted, small myelinated fibers (x400). b Electron microscopic examination. It showed unmyelinated
axons with atrophy and vesicular changes. c SDH reaction of skeletal muscle. Scattered myofibers showed increased positive reaction (x200). d Myofibers
with focal subsarcolemmal accumulation of enlarged mitochondria
Fig. 5 Effect of knockdown of MPV17 and overexpression of mutant proteins on cell proliferation. a Confirmation of MPV17 knockdown in NSC34
by RT-PCR. b Inhibitory effect of the proliferation of NSC34 by the abrogation of MPV17 using specific siRNA. c Affection of MPV17 mutations on
cell viability against reactive oxygen species (ROS). Knockdown of MPV17 using specific siRNA renders NSC34, a mouse motor neuronal cell line,
more sensitive to H2O2 treatment (24 h). d Western blot analysis to determine the expression of mutant proteins. e Overexpression of mutant
proteins affected cell proliferation of NSC34. f Changes in the mitochondrial OXPHOS system. Western blotting using OXPHOS detection cocktail
antibody was performed after overexpression of wild-type and mutant MPV17 proteins. For cell proliferation and survival assay, 4–6 wells per each
sample were counted. Data are presented as mean ± SEM. Statistical analysis were performed using Student’s t-test. * and #, p < 0.05; **and ##,
p < 0.01. Statistical significance was determined either with control myc (* and **) or wild-type MPV17 (# and ##) in (e)
shown). We observed that MPV17 abrogation significantly
affects the mitochondrial OXPHOS system, susceptibility
to ROS, and cell proliferation. These data suggest that
sustained expression of MPV17 is critical to PNS.
To address the effect of the p.R41Q mutation, we
compared the effect of the mutant protein on cell
viability with several previously reported mutants.
Structurally, p.R41Q mutation is similar to p.R50Q in that both
amino acids are located in the matrix region between
the first two transmembrane regions [1, 2, 18]. However,
the clinical phenotype is quite different [3, 14]. In
addition, the phenotype of a compound heterozygote
mutation, p.KM88-89ML and p.L143*, is closest to that
of the present mutation, although the patient exhibited a
fatty liver and hearing loss . Analysis of the cell
proliferation revealed that overexpression of the present
mutation exhibited a negative effect, similar to p.R50Q
and p.L143* mutation. In addition, several mutant
proteins mildly affected the mitochondrial OXPHOS
system. The mitochondrial oxidative phosphorylation
system was affected by the expression of mutant
proteins or partly by wild-type MPV17. This result is
consistent with a previous report that liver samples, from
MPV17 mutation (P64R) harboring patients, revealed
low levels of complex I, III and IV subunits. Although
we could not determine the effect on the integrity of
mitochondrial DNA (mtDNA) using patients’ samples,
we tried with overexpression model. There was
no mtDNA deletion, however, we observed that
overexpression of wild-type or mutant MPV17 induced mtDNA
depletion (Additional file 2: Figure S1b). These data
suggest that overexpression of MPV17 might cause
Recently, Uusimaa et al. reported two cases of a new
mutation at the same amino acid (p.R41W) in MPV17 .
The phenotype of the patient was milder than the
aforementioned patients who exhibited progressive,
neurological deterioration. They did not exhibited any defect in
muscle, liver cirrhosis nor focal fibrosis. Experimentally,
we observed that overexpression of R41W mutant in
NSC34 cell also affects cellular proliferation (Additional
file 2: Figure S1c). Thus these data implicate that mutation
at Arg41 predominantly cause peripheral neuropathy.
We suggest that a novel homozygous p.R41Q mutation in
MPV17 causes axonal sensorimotor polyneuropathy
without hepatoencephalopathy. Our observations will expand
the clinical spectrum of MPV17-causing disease and
suggest that MPV17 should be considered in screening tests
for patients presenting only with axonal sensorimotor
Additional file 1: Table S1. List of primers and siRNAs. Table S2.
Summary of exome sequencing data. Table S3. List of CMT- and
MTDPSrelated genes. Table S4. Polymorphic nonsynonymous variants in peripheral
neuropathy- and mitochondrial DNA depletion syndrome- related genes from
the exome date. (DOCX 43 kb)
Additional file 2: Figure S1. (a) Changes in the mitochondrial OXPHOS
system. Western blotting using OXPHOS detection cocktail antibody was
performed after knockdown of MPV17. Data are presented as mean ± SEM.
*, p < 0.05; **, p < 0.01. (b) Mitochondrial DNA (mtDNA) depletion assay.
HEK293 cells were treated with control (pCMV-myc), wild-type or mutant
MPV17 transcripts for 72 days, then total DNA was purified and mtDNA
depletion was analyzed by comparing the ratio of genomic DNA (beta
actin) and mtDNA (ND1 and COX1) using realtime PCR. (c) Overexpression
of mutant proteins affected cell proliferation of NSC34. Both MPV17 mutant
proteins, R41Q and R41W, affected cell proliferation. (TIFF 2825 kb)
MTDPS6: Mitochondrial DNA depletion syndrome 6; NNH: Navajo
neurohepatopathy; WES: Whole exome sequencing; OXPHOS: Oxidative
phosphorylation; mtDNA: Mitochondrial DNA; CMT: Charcot-Marie-Tooth
disease; CMT2A: Charcot-Marie-Tooth disease type 2A; MRC: Medical
research council; MCVs: Motor conduction velocities; CMAPs: Compound
muscle action potentials; SCVs: Sensory conduction velocities;
SNAP: Sensory nerve action potential; NADH-RT: NADH-tetrazolium
reductase; SDH: Succinate dehydrogenase; PAS: Periodic acid Schiff;
SNP: Single nucleotide polymorphism; MTT:
3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide; VEP: Visual evoked potential;
BAEP: Brainstem auditory evoked potential; MRI: Magnetic resonance imaging.
The authors declare no conflict of interest.
KWC and BOC conceived and coordinated the study. SBK, HJP, HK and
JL performed clinical assessments and data analysis. YRC, YBH, JHL, YJK,
HK, JSL, NJ, and SYW performed genetic and biochemical studies. YBH
and SCJ analyzed the data and prepared the manuscript. All authors
read and approved the final manuscript.
This study was supported by the Korean Health Technology R&D Project,
Ministry of Health & Welfare, Republic of Korea (HI12C0135 and HI14C3484)
and by the National Research Foundation of Korea (NRF) grants funded by
the Korean government, MSIP (NRF-2014R1A2A2A01004240).
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