Please cite this article in press as: Miyake et al., Biallelic TBCD Mutations Cause Early-Onset Neurodegenerative Encephalopathy, The American Journal of Human Genetics (2016), http://dx.doi.org/10.1016/j.ajhg.2016.08.005

REPORT
Biallelic TBCD Mutations Cause Early-Onset
Neurodegenerative Encephalopathy
Noriko Miyake,1,* Ryoko Fukai,1,2 Chihiro Ohba,1,2 Takahiro Chihara,3 Masayuki Miura,4,5
Hiroshi Shimizu,6 Akiyoshi Kakita,6 Eri Imagawa,1 Masaaki Shiina,7 Kazuhiro Ogata,7
Jiu Okuno-Yuguchi,8 Noboru Fueki,9 Yoshifumi Ogiso,10 Hiroshi Suzumura,11 Yoshiyuki Watabe,11George Imataka,11 Huey Yin Leong,12 Aviva Fattal-Valevski,13 Uri Kramer,13 Satoko Miyatake,1Mitsuhiro Kato,14 Nobuhiko Okamoto,15 Yoshinori Sato,16 Satomi Mitsuhashi,17,18 Ichizo Nishino,17,18
Naofumi Kaneko,19 Akira Nishiyama,19 Tomohiko Tamura,19 Takeshi Mizuguchi,1 Mitsuko Nakashima,1Fumiaki Tanaka,2 Hirotomo Saitsu,20 and Naomichi Matsumoto1,*
We describe four families with affected siblings showing unique clinical features: early-onset (before 1 year of age) progressive diffuse
brain atrophy with regression, postnatal microcephaly, postnatal growth retardation, muscle weakness/atrophy, and respiratory failure.
By whole-exome sequencing, we identified biallelic TBCD mutations in eight affected individuals from the four families. TBCD encodes
TBCD (tubulin folding co-factor D), which is one of five tubulin-specific chaperones playing a pivotal role in microtubule assembly in allcells. A total of seven mutations were found: five missense mutations, one nonsense, and one splice site mutation resulting in a frameshift. In vitro cell experiments revealed the impaired binding between most mutant TBCD proteins and ARL2, TBCE, and b-tubulin. The
in vivo experiments using olfactory projection neurons in Drosophila melanogaster indicated that the TBCD mutations caused loss offunction. The wide range of clinical severity seen in this neurodegenerative encephalopathy may result from the residual function ofmutant TBCD proteins. Furthermore, the autopsied brain from one deceased individual showed characteristic neurodegenerative findings: cactus and somatic sprout formations in the residual Purkinje cells in the cerebellum, which are also seen in some diseases associated with mitochondrial impairment. Defects of microtubule formation caused by TBCD mutations may underlie the pathomechanismof this neurodegenerative encephalopathy.

Microtubules, cylindrical structures composed of rows of
a/b-tubulin heterodimers, are essential components of all
eukaryotic cells and are involved in a number of functions
such as cell division, morphology, polarization, migration,and intracellular transport.1,2 Their unique feature of
growth (polymerization) and shrinkage (depolymerization) is known as microtubule dynamics.3,4 a/b-tubulin
heterodimers are formed with the aid of tubulin-specificchaperones comprised of five tubulin folding co-factors(TBC): TBCA, TBCB, TBCC, TBCD, and TBCE.5,6 a- andb-tubulin monomers initially bind with TBCB and TBCA,
respectively, and are passed to TBCE and TBCD, respectively, and form complexes together with TBCC and
ARL2.5,7–9
Of the five TBC genes, only mutations in TBCE(GenBank: NM_003193.4; MIM: 604934) have been foundto be associated with an autosomal-recessive hypoparathyroidism-retardation-dysmorphism syndrome (MIM:
241410).10 This is characterized by congenital hypoparathyroidism, microcephaly, hypoplasia of the corpus callosum, intellectual disability, growth failure, and dysmorphic features.11 It is also associated with a similar disease
with bone dysplasia, Kenny-Caffey syndrome (MIM:
244460).10 Here, we report biallelic mutations in TBCD(GenBank: NM_005993.4; MIM: 604649) encoding TBCD
and the resulting mutant phenotypes in humans.
In this study, eight affected individuals from two Japanese families, a Chinese family, and an Israeli family
were investigated (Figure 1A). The Israeli family was
consanguineous, but the others were not. Genomic DNA
was extracted from peripheral leukocytes using QuickGene-610L (Fujifilm) or formalin-fixed paraffin-embedded

1
Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan; 2Department of Neurology and
Stroke Medicine, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan; 3Department of Biological Science, Graduate Schoolof Science, Hiroshima University, Hiroshima 739-8526, Japan; 4Department of Genetics, Graduate School of Pharmaceutical Sciences, The University ofTokyo, Tokyo 113-0033, Japan; 5Agency for Medical Research and Development-Core Research for Evolutional Medical Science and Technology (AMEDCREST), Japan Agency for Medical Research and Development, Tokyo 100-0004, Japan; 6Department of Pathology, Brain Research Institute, Universityof Niigata, Niigata 951-8585, Japan; 7Department of Biochemistry, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan;
8Department of Neuropediatrics, Nagano Children’s Hospital, Azumino 399-8205, Japan; 9Division of Rehabilitation, Nagano Children’s Hospital, Azumino 399-8205, Japan; 10Department of Clinical Pathology, Nagano Children’s Hospital, Azumino 399-8288, Japan; 11Department of Pediatrics, Dokkyo
Medical University School of Medicine, Tochigi 321-0293, Japan; 12Genetics Department, Hospital Kuala Lumpur, Kuala Lumpur 50586, Malaysia; 13Pediatric Neurology Unit, Tel Aviv Sourasky Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel; 14Department of Pediatrics, Showa University School of Medicine, Tokyo 142-8666, Japan; 15Department of Medical Genetics, Osaka Medical Center and Research Institute forMaternal and Child Health, Osaka 594-1101, Japan; 16Department of Molecular Biology, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan; 17Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo
187-8502, Japan; 18Department of Medical Genome Development, Medical Genome Center, National Center of Neurology and Psychiatry, Tokyo 1878551, Japan; 19Department of Immunology, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan; 20Department ofBiochemistry, Hamamatsu University School of Medicine, Hamamatsu 431-3192, Japan*Correspondence: nmiyake@yokohama-cu.ac.jp (N. Miyake), naomat@yokohama-cu.ac.jp (N. Matsumoto)
http://dx.doi.org/10.1016/j.ajhg.2016.08.005.
Ó 2016 American Society of Human Genetics.

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Please cite this article in press as: Miyake et al., Biallelic TBCD Mutations Cause Early-Onset Neurodegenerative Encephalopathy, The American Journal of Human Genetics (2016), http://dx.doi.org/10.1016/j.ajhg.2016.08.005

Figure 1. Familial Pedigrees and TBCD
Mutations(A) The pedigrees of four families with biallelic TBCD mutations. The mutations areshown in red. Affected and unaffectedindividuals are shown as black and white
symbols, respectively. Affected individual
identities are as follows: individuals 1 (II-1
in family 1), 2 (II-2 in family 1), 3 (II-1 infamily 2), 4 (II-2 in family 2), 5 (II-1 in family3), 6 (II-2 in family 3), 7 (II-1 in family 4), and
8 (II-2 in family 4).
(B) TBCD cDNA and TBCD protein structures. The p.Met387Arg and p.Arg772Cys
variants fall within the SCOP domain, andp.Ala921Thr and p.Pro937Arg within the
TFCD_C domain. The seven variants foundin the cDNA and protein are indicated by arrows. Functional domains of the TBCD protein were predicted by the SMART program.
(C) The evolutionarily conserved aminoacids in the missense variants. The conserved
amino residues are marked in red.
(D) The facial features of the affected individuals. From left to right, individual 1 at
3 months, individual 2 at 3 years, individual
3 at 4 months, individual 4 at 3 months, individual 5 at 1 year, and individual 6 at 1 year.

brain tissue using QIAamp DNA FFPE tissue kit (QIAGEN)(for individual 1: II-1 in family 1 in Figure 1A) according
to the manufacturer’s instructions. Blood samples or brain
tissue from affected individuals and their families werecollected after obtaining written informed consent. The
study protocol was approved by the institutional review
board of Yokohama City University School of Medicine.
To identify the genetic cause of the disease, we performed
whole-exome sequencing as described previously.12 The
presence of affected siblings in the families implies anautosomal-recessive mode of inheritance (Figure 1A).
Therefore, we focused on genes with homozygous or compound heterozygous variants and identified biallelic TBCD
mutations in all probands in the four families (Figure 1A
and Tables S1–S3). Using Sanger sequencing, we confirmedthat the biallelic mutations perfectly co-segregated withthe disease status (Figure S1). A total of seven mutations
were found in TBCD: five missense, one nonsense, and asplicing mutation (Table 1). These variants were unreported or extremely rare (%0.0002) in ExAC, EVS, 1000

2

Genome, HGVD, and our in-house
exome database (n ¼ 575). All of themissense variants were predicted to bepathogenic by PolyPhen-2 and MutationTaster. In addition, four alteredamino residues due to missense mutations were located within the functional
domains (Figure 1B). Five of the amino
residues altered by the missense mutations were evolutionally conserved(four from zebrafish to human and one
from mouse to human) (Figure 1C).
The intronic mutation (c.156412C>G) resulted in an
11-bp insertion of intronic sequence between exons 16
and 17, which was confirmed at the cDNA level to causea frameshift (p.Gly522Phefs*14) subjected to nonsensemediated mRNA decay (Figure S2). This genetic evidence
strongly indicates that biallelic TBCD mutations may cause
this neurodegenerative encephalopathy.
The clinical data of all affected individuals are shown in
Table 2 and the Supplemental Note. In brief, affectedindividuals with biallelic TBCD mutations showed anearly onset of symptoms (before 1 year of age). The firstsymptoms varied among the families but were similar inaffected siblings from the same family: neonatal asphyxia
in family 1, decreased movement and hypotonia at
1 month of age in family 2 and at 5 months in family 3,
and seizures at 9–11 months in family 4. The affected individuals commonly had progressive clinical courses withmuscle weakness, absent visual tracking, and postnatal
microcephaly. More than half of the affected individuals
had postnatal growth failure, seizure, respiratory failure,

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Please cite this article in press as: Miyake et al., Biallelic TBCD Mutations Cause Early-Onset Neurodegenerative Encephalopathy, The American Journal of Human Genetics (2016), http://dx.doi.org/10.1016/j.ajhg.2016.08.005
Table 1.

Summary of TBCD Mutations

Mutation

Amino Acid
ChangePosition on
chr17 (hg19) dbSNP141

PolyPhen-2 MutationTaster(Score)(Score)ExAC

1000
In-house
EVS Genomes HGVD (n ¼ 575)

c.1160T>G

p.Met387Arg

80,767,595

nopossiblydamaging(0.733)disease causing(0.996)

no

no

no

no

0

c.156412C>G p. Gly522Phefs*14 80,851,411

no

NA

NA

no

no

no

no

0

c.2280C>A

p.Tyr760*

80,882,834

noNA

disease causing(1.000)

no

no

no

no

0

c.2314C>Tp.Arg772Cys

80,882,868

rs181969865 probably
damaging(0.972)disease causing(1.000)nono

0.0002

no0

c.2761G>A

p. Ala921Thr

80,887,056

noprobablydamaging(1.000)disease causing(1.000)

no

no

no

no

0

c.2810C>G

p.Pro937Arg

80,887,105

noprobablydamaging(1.000)disease causing(1.000)

no

no

no

no

0

c.3365C>T

p.Pro1122Leu

80,896,008

noprobablydamaging(1.000)disease causing(1.000)0.00003645 no

no

no

0

The description of the mutation was based on GenBank: NM_005993.4. Abbreviations are as follows: ExAC, Exome Aggregation Consortium; EVS, Exome Variant
Server; HGVD, Human Genetic Variation Database; NA, not applicable. Data of minor allele frequencies were examined in May 2016.

regression, optic nerve atrophy, hypotonia, and muscle atrophy. In addition, two affected individuals had variableblood pressure and two other individuals showed urinary
incontinence, one of whom also had fecal incontinence,suggesting an involvement of the autonomic system insome affected individuals. Six affected individuals in threeof the families became immobile and five required respiratory management. Three of the eight affected individuals
were deceased by 20 years of age. Most affected individuals
had muscle weakness and atrophy. A muscle biopsy at
12 days of age (corrected gestational age: 5 days) in individual 2 (family 1, II-2) revealed the following myopathic
changes: (1) variation in moderate muscle fiber size,
ranging from 5 to 15 mm in diameter (Figure 2A); (2)numerous type 2C fibers; (3) atrophy of type 1 muscle fibers (Figure 2B and 2C); and (4) dot-like staining in the
myofiber cytoplasm after the NADH-tetrazolium reductase
reaction (Figure 2D). The neuromuscular junction was notincluded in the section. No evidence of neuropathy wasobserved. These findings may reflect the immaturity ofmuscle fibers. However, it was not possible to confirm
this because only limited samples were available. Interestingly, four affected individuals had episodes of elevatedcreatine kinase (CK). The elevation of CK was noticed
only at birth (individual 1, 1,338 IU/L; individual 2,
1,345 IU/L) in family 1, and at several time points in individual 3 (max. 285 IU/L at 7 months) and individual 4(max. 752 IU/L at 11 months) (Table 2, Supplemental
Note). We believe that elevated CK was caused by asphyxia
and bone fracture in family 1 and by frequent episodes ofafebrile seizures in family 2. However, the actual triggers
remain unclear. Brain imaging studies from three affectedindividuals indicated progressive brain atrophy involving
the cerebrum, corpus callosum, cerebellum, and brain
stem (Figures 3 and S3). As early as 3 months of age, atrophy in the frontal lobe and enlarged ventricles (especiallyin the frontal horn of the lateral ventricles) were detected.
Despite the flat and expressionless faces, downturned corners of the mouth, and open mouths, we did not see any
specific dysmorphic features (Figures 1D and S4).
Interestingly, some clinical features overlapped between
biallelic TBCD and TBCE mutations: early-onset (<1 year)
intellectual disability, postnatal growth failure, hypoplastic corpus callosum, and microcephaly are common inboth diseases, while intrauterine growth retardation andhypoparathyroidism were not seen in individuals with
TBCD mutations (Table S4).11,13
TaqMan assays revealed that TBCD was ubiquitously expressed in all tissues, but highly expressed in brain, heart,and skeletal muscle in the fetus and adult (Figure S5). The
expression pattern was consistent with the affected organs,including the central nervous system and skeletal muscles,in affected individuals with TBCD mutations.
An autopsy was performed on individual 1 (family 1,II-1). Although no malformation of the internal organs
was detected (Table S5), striated muscle volume, including
the iliopsoas, was severely decreased and the diaphragm
was extremely thin. The brain and spinal cord were generally atrophic. In the cerebrum, there was severe atrophy ofthe cortex and white matter and the lateral ventricles weremarkedly dilated (Figure 2E). The corpus callosum was
rudimentary, although the basal ganglia and thalamus
were generally well formed. Both the anterior and posteriorlimbs of the internal capsules were severely atrophic.

The American Journal of Human Genetics 99, 1–12, October 6, 2016 3

4Genetic and Clinical Information of the Affected Individuals with TBCD Mutations

Family ID

Family 1

Family 2

Family 3

Family 4

Ethnicity

JapaneseJapanese

ChineseIsraelMutations andVariations

c.156412C>G (splicing), c.2314C>T(p.Arg772Cys)

c.1160T>G (p.Met387Arg),
c.2761G>A (p. Ala921Thr)

c.2280C>A (p.Tyr760*), c.3365C>T(p.Pro1122Leu)

c.2810C>G (p.Pro937Arg), c.2810C>G(p.Pro937Arg)

Sex

femalemale

femalemale

male

femalefemalefemaleIndividual ID

II-1

II-2

II-1

II-2

II-1

II-2

II-1

II-2

Individual
numberindividual 1

individual 2

individual 3

individual 4

individual 5

individual 6

individual 7

individual 8

Pregnancy

hydramnion

hydramnion

normalnormalnormalnormalnormalnormalAge of onset

at birth

at birth

1 month

1 month

5 months

5 months

11 months

9 months

Initialsymptom(s)asphyxia, bone fracture

asphyxia, bone fracture

paucity of
movement

hypotonia

hypotonia, regression

hypotonia, regression febrile seizure

generalized seizure

Age of evaluation 4 years

19 years

9 years

5 years

2 years

3 years

17 years

16 years

Growth failurea

yes

yes

yes

yes

yes

yes

no

no

Seizurenono

West syndrome cataplexy

GTS

GTS

GTS/GTCS

GTS/GTCS

Onset age of
seizureND

ND

4 months

6 months

6 months

1 years

11 months

9 months

Respiratory
failure

yes

yes

yes

yes

yes

no

no

no

Regression

ND

ND

yes

yes

yes

yes

yes

yes

Visual tracking

no

no

no

no

no

no

no

no

Optic nerve
atrophy

no

yes

yes

yes

yes

no

no

no

Postnatal
microcephaly

yes

yes

yes

yes

yes

yes

yes

yes

Ataxia

NDb

NDb

yes

yes

no

no

no

no

Hypotonia

yes

yes

yes

yes

yes

yes

no

no

yes

yes

yes

c

c

yes

yes

b

Muscle weakness

yes

ND

Muscle atrophy

yes

yes

yes

yes

no

no

yes

yes

no

no

yes

yes

no

no

no

no

no

no

no

no

yes

no

no

no

no

no

yes

no

Fasciculationd

Urinaryincontinence

ND

yes

Fecal
incontinence

no

no

e

yes

(Continued on next page)

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The American Journal of Human Genetics 99, 1–12, October 6, 2016

Table 2.

yes

Abbreviations are as follows: ND, no data; GTS, generalized tonic seizure; GTCS, generalized tonic clonic seizure; CK, creatine kinase.
Judged as height < 2 SD.
Not assessed because individual was immobile.
c
Floppy and almost immobile.
dFasciculation in tongue.
e
Neurogenic bladder.
b

a

no

walking, moderate
walking, moderateintellectual disability intellectual disability

no
no
no
yes

Prognosis

yes
yes

immobile with ventilation immobile with ventilation immobile withand deceased at 4 years
and deceased at 19 years
ventilation(respiratory failure)(anaphylactic reaction)

Elevation of CK

immobile with immobile with ventilation immobile
ventilationand deceased at 4 years(pneumonia)

no
no
ND
ND
no

moderate
ND

moderatenono

Blood-pressure
variability

Table 2.

Continued

no

no
no
Disturbed
sweating

Family 2
Family 1
Family IDnoFamily 3noFamily 4no

Please cite this article in press as: Miyake et al., Biallelic TBCD Mutations Cause Early-Onset Neurodegenerative Encephalopathy, The American Journal of Human Genetics (2016), http://dx.doi.org/10.1016/j.ajhg.2016.08.005

Histopathological examination revealed systemic laminar
necrosis in the cerebral cortex, although a normal sixlayer structure was observed with no cytologicallyabnormal neurons, such as dysmorphic neurons orballoon cells (Figure 2F). Furthermore, the hippocampus,
thalamus, pontine nucleus (Figure 2G), globus pallidus,
and inferior olivary nucleus had severe neuronal loss andgliosis. Many globular structures of unknown origin wereobserved in the internal segment of the globus pallidus.
Purkinje and granule cells in the cerebellum were severely
depleted.
These histological abnormalities might be ascribable, atleast in part, to intrapartum asphyxia, in which the patternof neuronal injury may be almost comparable to that of‘‘full-term’’ severe asphyxia.14 However, in this individual,
other anatomical regions susceptible to asphyxial injury interm infants, including the putamen (Figure 2H), quadrigeminal bodies, substantia nigra (Figure 2I), and dorsal vagal
nucleus, had only mild gliosis without apparent neuron
loss. Additionally, in the cerebellum, many residual Purkinje cells had focal swellings of the dendrites (cactus/
asteroid bodies) (Figure 2J) and some had halo-like amorphous structures around the cell bodies (somatic sprouts)(Figure 2K). Neuronal depletion was severe in the dentate
nucleus (Figure 2L). In addition, the brainstem lower motor neuron (LMN) nuclei, including the oculomotor andhypoglossal nuclei, had severe neuronal loss and associated gliosis. In the spinal cord, the lateral corticospinal
tracts and dorsal column showed severe degeneration
with myelin pallor (Figure 2M). The anterior horns showed
severe loss of the LMNs and associated gliosis, which wassomewhat more apparent in the lateral nuclear group(Figure 2N). The spinal anterior roots were severely
atrophic, whereas the posterior roots were preserved. The
diaphragm was severely degenerated with fibrosis andscattered pyknotic nuclear clumps (Figure 2O).
We constructed homology-modeled structures of human
TBCD using the Protein Homology/analogY Recognition
Engine v.2.0 (Phyre2) server,15 because no experimental
structure is available. The resultant models had a-solenoid
repeats with pairs of antiparallel alpha helices, which havea deformable nature. We also employed an in silico detector of an a-solenoid repeat based on an amino acidsequence, Alpha-rod Repeat Detector 2 (ARD2)16 to verify
the modeled structures. The results by ARD2 were consistent with those by the Phyre2 prediction (data not shown).
In the structures obtained, the side chains of Met387 andAla921 are buried in the hydrophobic cores of a solenoid
repeat (Figure S6A). Therefore, the p.Met387Arg andp.Ala921Thr variants might affect the folding of the a-solenoid repeat. The methylene part of the Arg772 side chain ispart of a hydrophobic core and the guanidium group is
exposed to the protein surface in the modeled structure(Figure S6A). Thus, the p.Arg772Cys variant might also
affect at least the folding structure. Because Pro937 andPro1122 were predicted to reside within a loop between
antiparallel a helices and be exposed to a protein surface

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Please cite this article in press as: Miyake et al., Biallelic TBCD Mutations Cause Early-Onset Neurodegenerative Encephalopathy, The American Journal of Human Genetics (2016), http://dx.doi.org/10.1016/j.ajhg.2016.08.005

Figure 2. Histopathological Findings in an Individual with Biallelic TBCD Mutations(A–D) Histology of a muscle biopsy from individual 2 at the age of 12 days (corrected gestational age: 5 days). Shown are (A) hematoxylin
and eosin staining, (B) myosin ATPase (pH 4.2), (C) myosin ATPase (pH 10.5), and (D) NADH-tetrazolium reductase. The arrows indicate
type 1 fibers. Scale bars represent 20 mm.
(E) The cerebral cortex of individual 1 showing systemic laminar necrosis, which is more marked in the temporal lobe. The white matteris also atrophic and associated with marked dilatation of the lateral ventricles. The corpus callosum (arrowhead) and posterior limb of theinternal capsule (arrows) are thin. The basal ganglia and thalamus are generally well formed.
(F) The frontal cortex has a normal six-layer architecture. Laminar necrosis is evident in the middle cortical layers.
(legend continued on next page)6

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Please cite this article in press as: Miyake et al., Biallelic TBCD Mutations Cause Early-Onset Neurodegenerative Encephalopathy, The American Journal of Human Genetics (2016), http://dx.doi.org/10.1016/j.ajhg.2016.08.005(Figure S6A), the p.Pro937Arg and p.Pro1122Leu variants
might affect the loop structure and/or interactions withother molecules, although no functional data are available.
TBCD functions as a component of the tubulin chaperone
multiprotein complex (Figure S6B).17 Therefore, all of thevariants described here have the potential to impair complex formation.
Next, we examined protein-protein interactions between TBCD (wild-type and mutant) and other complex
components (a/b-tubulin, TBCE, TBCC, and ARL2) by coimmunoprecipitation. The open reading frames of human
TBCD, TBCE, TBCC (GenBank: NM_003192.2), and ARL2
(GenBank: NM_001667.3) were amplified using human
cDNA as a template purchased from Clontech. The PCR
products were cloned into the Gateway pDONR221 vector(Life Technologies) to create the entry clones, and eachsequence was confirmed by Sanger sequencing. The QuickChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies) was used to create mutant constructs. To create
the transient mammalian expression vectors, each entry
clone was used for LR recombination with pcDNA3.1/
nV5-DEST (for N0 V5-tagged TBCC or TBCE), pcDNADEST53 (for N0 GFP-fused TBCD), or pcDNA-DEST40 (forC0 V5-6xHis-tagged ARL2). Wild-type TBCD interact with
ARL2, TBCE, and b-tubulin (Figure 4). No apparent binding
was observed between TBCD and a-tubulin or TBCD and
TBCC (data not shown); therefore, we focused on TBCD
binding with ARL2, TBCE, and b-tubulin. As for thebinding with ARL2, TBCD mutants p.Met387Arg almost
completely, and p.Arg772Cys, p.Ala921Thr, p.Pro1122Leu,
and p.Tyr760* partially, lost binding capacity comparedwith wild-type TBCD. p.Pro937Arg retained its binding capacity (Figure 4A). Mutant TBCD showed similar impaired
binding to TBCE (Figure 4B). All TBCD mutants showeddecreased binding to endogenous b-tubulin (Figure 4C).
These experiments suggest that all the investigated TBCD
mutations may impair the proper formation of microtubule chaperone complexes.
Finally, to investigate the possible effects of disease-associated TBCD mutations in vivo, we took advantage ofmosaic analysis with a repressible cell marker (MARCM)
in Drosophila, which allowed us to analyze gene function
in vivo at single-cell resolution.18 We expressed mem-brane-tethered GFP (mouse CD8::GFP, mCD8-GFP) to visualize the neuronal morphology of olfactory projection
neurons (PNs) in the intact brain (Figure 5).19,20 The axonsof DL1 PNs elongate and form axonal branches at the calyxof the mushroom body and the lateral horn. The dendrites
of PNs target a single glomerulus (DL1 glomerulus) in theantennal lobe, the first olfactory center in the Drosophilabrain (Figures 5A and S7 for wild-type axon and dendrites,
respectively). As we have previously reported,21 PNs homozygous for tbcd1 mutations (tbcd1 PNs), in an otherwise
largely heterozygous animal, had shorter axons and/or
reduced axonal branches (Figures 5B–5D). Next, we carriedout a MARCM-rescue experiment, in which we cell autonomously expressed cDNA of wild-type human TBCD intbcd1 PNs and found that overexpression of human
TBCD significantly suppressed the axonal phenotypes of
tbcd1 PNs (Figure 5E). In addition, the dendritic phenotypeof tbcd1 PNs also tended to be suppressed by human TBCD
overexpression (Figure S7). These results suggest that thefunction of TBCD in neuronal morphogenesis is partlyconserved between Drosophila and humans.
To test the effect of disease-associated TBCD mutations,
we performed MARCM-rescue experiments with human
TBCD carrying disease-associated mutations. To avoid thepositional effect that causes unexpected transgene expression differences, we utilized the PhiC31-integrase system
to generate UAS transgenic animals and all the UAS transgenes were inserted in an attP2 landing site.22 Human
TBCD bearing a disease-associated mutation was less able
to suppress the axonal phenotype of tbcd1 PN comparedwith wild-type human TBCD (Figure 5E). A similar tendency was also observed in PN dendrites (Figure S7). These
results indicate that the TBCD disease-associated mutations we tested cause loss of function of the TBCD protein.
In this study, we have reported four families with earlyonset neurodegenerative encephalopathy associated withbiallelic TBCD mutations. Core clinical features include
early-onset progressive diffuse brain atrophy with regression, postnatal microcephaly, postnatal growth delay,
muscle weakness/atrophy, and respiratory failure. Ourin vitro and in vivo studies showed that seven TBCD mutations are likely to lead to loss of function of the TBCD protein. Interestingly, the clinical course was very similar(G) The pontine nucleus with severe neuronal loss and gliosis. Note that both the longitudinal and transverse fibers are degenerative, andlongitudinal fibers are almost unrecognizable.
(H) The putamen has a normal composition of medium spiny and large aspiny neurons (arrows). The putamino-pallidal fibers areevident (arrowheads).
(I) Substantia nigra is well preserved in this individual.
(J–L) Loss of Purkinje and granule cells is evident in the cerebellum. The residual Purkinje cells show torpedoes (J, arrows) and asteroid
bodies/cactus (J, arrowheads and inset), as well as halo-like amorphous structures (somatic sprouts) (K, arrowheads). Neuron loss is also
apparent in the dentate nucleus (L).
(M) Anterior horn volume is reduced in the lumbar cord. The anterior nerve roots are severely atrophic (arrows), whereas the posteriorroots are almost preserved. Degeneration of the lateral corticospinal tracts (arrowheads) and posterior column is also evident.
(N) The lumbar anterior horn with severe loss of the lower motor neurons and associated gliosis.
(O) Severe degeneration of the muscle fibers in the diaphragm, with fibrosis and scattered pyknotic nuclear clumps (arrow).
Shown are Klüver-Barrera staining (E, F, G, I, L, M), hematoxylin and eosin staining (H, J, N, O), and Bodian staining (K). Scale bars represent 1 cm (E), 500 mm (F), 200 mm (G, I), 100 mm (H, L, N, O), 160 mm (J), 50 mm (J inset and K), and 1.5 mm (M).

The American Journal of Human Genetics 99, 1–12, October 6, 2016 7

Please cite this article in press as: Miyake et al., Biallelic TBCD Mutations Cause Early-Onset Neurodegenerative Encephalopathy, The American Journal of Human Genetics (2016), http://dx.doi.org/10.1016/j.ajhg.2016.08.005

Figure 3. Neuroimaging of Affected Individuals with Biallelic TBCD Mutations(A) The neuroimages of individual 2 at
8 months (computed tomography: CT), 1
year 8 months and 16 years (T1-weighted
MRI). Severe, progressive enlargement ofbilateral ventricles, thin brain stems, andsevere cerebellar atrophy are shown.
(B) The axial brain paired T1- and T2weighted MRI images of individual 3 at
3 months and 7 months and CT at 4 years(right).
(C) The sagittal images of individual 3. T1weighted MRI at 3 months (left) and CT at
4 years (right). Thinned corpus callosumand enlargement of the great cistern can beseen on the MRI (left).
(D) The serial images from individual 4.
From upper left to upper right, axial T1weighted MRI and T2-weighted MRI at
5 months, CT at 1 year 6 months, and CT
at 2 years. Gyral pattern and myelination
seemed to be consistent with age, whereas
the progressive atrophy of the cerebral cortex (predominantly in the frontal cortex)
and enlargement of the ventricles is remarkable. From lower left to lower right, twosections of sagittal T1-weighted MRI at
5 months, sagittal and coronal sections
from CT at 2 years.
(E) T1- and T2-weighted MRI images of individual 4 at 5 months. The white arrowheads
indicate a chronic subdural hematoma.
(F) T1- and T2-weighted MRI images of individual 6 at 1 year 4 months.

within individual families, but differed between families.
This might be partly explained by the specific mutations
in each family. The siblings in family 4 with a homozygous missense mutation (c.2810C>G [p.Pro937Arg]) had
a milder phenotype, possibly because binding of thealtered TBCD protein with b-tubulin was only mildly
affected. In more severe clinical cases (such as the deceased
individuals in families 1 and 3), affected individuals carried truncating and missense variants that significantly
impaired TBCD binding to ARL2, TBCE, and b-tubulin(Table S6). This indicates that biallelic TBCD mutations
may lead to a wide range of clinical severities dependingon the level of residual protein function resulting fromthe specific TBCD mutation.

8

We neuropathologically examined
an autopsied individual (individual 1)
in whom the original lesions caused
by TBCD mutations could have beenmasked by asphyxial effects. However,
we found the following pathologicalfindings that could not be easily explained by asphyxia: (1) neuronal lossof dentate nuclei of the cerebellumand LMN in the brainstem and anterior
horn of the spinal code and (2) somatic
sprout and cactus formations in cerebellar Purkinje cells.
Neuronal loss in the dentate nuclei and LMN may also
be caused by asphyxia.23 However, regions that are known
to be susceptible to asphyxia, such as the putamen andquadrigeminal bodies,14 were well preserved in thisaffected individual. Therefore, the disproportionally severe
loss of LMNs in the brainstem, spinal cord, and the cerebellar dentate nucleus may arise from lesions caused by
TBCD mutations, rather than asphyxia. In addition, cactus
formations are usually observed in Menkes disease andmitochondrial diseases,24–26 and somatic sprouts havebeen reported in Menkes disease and spinocerebellar ataxia
type 31,27–29 but not in asphyxia. Notably, mitochondrial
dysfunction is also associated with Menkes disease.30

The American Journal of Human Genetics 99, 1–12, October 6, 2016

Please cite this article in press as: Miyake et al., Biallelic TBCD Mutations Cause Early-Onset Neurodegenerative Encephalopathy, The American Journal of Human Genetics (2016), http://dx.doi.org/10.1016/j.ajhg.2016.08.005

Mitochondria are essential organelles providing energy
to all eukaryotic cells. Most mitochondria are newly produced in the soma and are transported to the distal endof axons and dendrites.31–33 In neurons, mitochondria
are distributed unevenly through microtubule trafficking,depending on local energy demand.33–36 Microtubules
are essential for long-distance axonal transport,32,37 andimpaired mitochondrial transport results in neuronaldegeneration.33,38–40 Reduced levels of TBCD protein affectmicrotubule network formation and impair microtubuletransport in tbcd1 PNs.41 Furthermore, TBCD is also
required for axon and dendrite maintenance in PNs.41
Therefore, TBCD depletion may result in an abnormal
microtubule network and abnormal microtubule trafficking in the human brain. Consequently, energy supply
to neuronal cells is reduced, causing neuronal degradation.
The formation of cactus and somatic sprouts might reflectstructural and metabolic abnormalities resulting from disrupted mitochondrial transport in neuronal cells caused
by TBCD depletion.
In conclusion, we have described an early-onset neurodegenerative encephalopathy caused by biallelic TBCD
mutations. We performed in vitro and in vivo experiments which revealed that the described TBCD mutations
probably cause loss of function of the TBCD protein. We
also investigated a possible genotype-phenotype correlation, particularly with regard to disease severity. Our
knowledge of human TBCD mutations is currently
limited, and therefore more genetic and clinical studies
are required to better understand the clinical consequences of TBCD mutations.

Figure 4. Intermolecular Associations between TBCD and Its
Binding Partners(A and B) The molecular interaction between human TBCD and
ARL2 (A) or TBCE (B). GFP-fused wild-type TBCD (159 kDa) or
its mutants were transiently co-overexpressed with V5-6xHistagged ARL2 (22 kDa) or V5-tagged TBCE (60 kDa) in HEK293T

cells using the X-treme GENE9 DNA transfection kit (Roche).
At 48 hr after transfection, the cells were collected andlysed with lysis buffer (25 mM Tris-HCl [pH 7.5], 100 mM NaCl,
2 mM EDTA, 0.5% Triton X-100, and protease inhibitors).
GFP-fused TBCD proteins were immunoprecipitated using a
rabbit polyclonal anti-GFP antibody (Abcam cat# ab6556,
RRID: AB_305564) and Dynabeads Protein-G (Thermo Fisher
Scientific). Input and immunoprecipitated samples (IP) were
immunoblotted with mouse monoclonal anti-GFP (Roche) andanti-V5 antibodies (Thermo Fisher Scientific cat# R960-25, RRID:
AB_2556564).
(C) The binding of human TBCD and endogenous b-tubulin(50 kDa). Wild-type GFP-fused TBCD or its mutants were transiently overexpressed in HEK293T cells. GFP-fused TBCD proteins
were immunoprecipitated with a rabbit polyclonal anti-GFP
antibody and immunoblotted with mouse monoclonal anti-GFP
or anti b-tubulin antibodies (Chemicon International cat#
MAB3408). Horseradish peroxidase-conjugated goat anti-mouse
IgG antibody (Jackson ImmunoResearch) was used as thesecondary antibody. Immunodetection was performed using
Supersignal West Dura Extended Duration Substrate (Thermo
Fisher Scientific) on a ChemiDoc Touch Imaging system (BioRad Laboratories).
At least three independent experiments were performed toconfirm the consistency of results for TBCD binding with ARL2,
TBCE, or b-tubulin. Mock indicates only GFP protein (27 kDa).
‘‘þ’’ and ‘‘’’ denote the presence and absence of V5-tagged proteins (or only V5-tag with the size of 1 kDa, which could not be
detected due to its small molecular weight), respectively.

The American Journal of Human Genetics 99, 1–12, October 6, 2016 9

Please cite this article in press as: Miyake et al., Biallelic TBCD Mutations Cause Early-Onset Neurodegenerative Encephalopathy, The American Journal of Human Genetics (2016), http://dx.doi.org/10.1016/j.ajhg.2016.08.005

Figure 5. Human TBCD with Disease-Associated Mutations Impaired NeuronalMorphology(A–D) The representative axonal morphologies of Drosophila olfactory projection neurons. The axons of DL1 single-cell clones forwild-type Drosophila (A) show the typicalbranching pattern at the calyx of the mushroom body (MB) and lateral horn (LH).
Axons of tbcd1 Drosophila PNs show branching and elongation defects (B–D). The dorsal
branch at the LH is often affected (arrow
in A–C). Axons cannot reach the MB in thesevere class (no axon is visible in D). PN
morphologies and Bruchpilot (presynaptic
marker) are shown in green (anti-CD8 antibody) and magenta (nc82), respectively.
Scale bars represent 25 mm.
(E) Quantification of MARCM-rescue experiments. Each gray scale corresponds to thecolors in (A)–(D) above. n indicates thenumber of MARCM clones examined.
Because the number of clones we canobserve in the Drosophila brain cannot be
controlled, we analyzed a variable numberof clones (minimum n ¼ 10). Details forthe generation of transgenic flies, MARCM
clone induction, immunohistochemistry of
fly brains, image processing, and quantification methods are available upon request.

Supplemental Data

Web Resources

Supplemental Data include supplemental note, seven figures, andsix tables and can be found with this article online at http://dx.
doi.org/10.1016/j.ajhg.2016.08.005.

ExAC Browser (accessed May 2016), http://exac.broadinstitute.org/
GATK, https://www.broadinstitute.org/gatk/
GenBank, http://www.ncbi.nlm.nih.gov/genbank/
Human Genetic Variation Database (HGVD), http://www.genome.
med.kyoto-u.ac.jp/SnpDB/
NHLBI Exome Sequencing Project (ESP) Exome Variant Server,
http://evs.gs.washington.edu/EVS/
Novoalign, http://www.novocraft.com/main/page.php?s¼novoalignOMIM, http://www.omim.org/
Picard, http://broadinstitute.github.io/picard/
RRID, https://scicrunch.org/resources
SMART, http://www.smart.embl-heidelberg.de/Acknowledgments
The authors appreciate the participation of all the affected individuals and their families in this study. This work was supported by
grants for Research on Measures for Intractable Diseases; Comprehensive Research on Disability Health and Welfare, the Strategic
Research Program for Brain Science; Initiative on Rare and Undiagnosed Diseases in Pediatrics and Initiative on Rare and Undiagnosed Diseases for Adults from the Japan Agency for Medical
Research and Development; Grants-in-Aid for Scientific Researchon Innovative Areas (Transcription Cycle and Comprehensive
Brain Science Network) from the Ministry of Education, Science,Sports and Culture of Japan; Grants-in-Aid for Scientific Research(B and C) and Challenging Exploratory Research from the Japan
Society for the Promotion of Science; Creation of Innovation Centers for Advanced Interdisciplinary Research Areas Program in the
Project for Developing Innovation Systems from the Japan Scienceand Technology Agency; the Takeda Science Foundation; the Yokohama Foundation for Advancement of Medical Science; andthe Hayashi Memorial Foundation for Female Natural Scientists.
Received: May 16, 2016
Accepted: August 9, 2016
Published: September 22, 2016

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