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ARTICLES
Anna-Kaisa Anttonen,
MD, PhD*
Taru Hilander, MSc*
Tarja Linnankivi, MD,
PhD
Pirjo Isohanni, MD, PhD
Rachel L. French, BSc
Yuchen Liu, PhD
Miljan Simonovic, PhD
Dieter Söll, PhD
Mirja Somer, MD, PhD
Dorota Muth-Pawlak,
PhD
Garry L. Corthals, PhD
Anni Laari, MSc
Emil Ylikallio, MD, PhD
Marja Lähde, MD
Leena Valanne, MD, PhD
Tuula Lönnqvist, MD,
PhD
Helena Pihko, MD, PhD
Anders Paetau, MD, PhD
Anna-Elina Lehesjoki,
MD, PhD
Anu Suomalainen, MD,
PhD
Henna Tyynismaa, PhD
Correspondence to
Dr. Tyynismaa:
henna.tyynismaa@helsinki.fi
Supplemental data
at Neurology.org
Selenoprotein biosynthesis defect causes
progressive encephalopathy with elevated
lactate
ABSTRACT
Objective: We aimed to decipher the molecular genetic basis of disease in a cohort of children with
a uniform clinical presentation of neonatal irritability, spastic or dystonic quadriplegia, virtually
absent psychomotor development, axonal neuropathy, and elevated blood/CSF lactate.
Methods: We performed whole-exome sequencing of blood DNA from the index patients. De-
tected compound heterozygous mutations were confirmed by Sanger sequencing. Structural pre-
dictions and a bacterial activity assay were performed to evaluate the functional consequences of
the mutations. Mass spectrometry, Western blotting, and protein oxidation detection were used
to analyze the effects of selenoprotein deficiency.
Results: Neuropathology indicated laminar necrosis and severe loss of myelin, with neuron loss
and astrogliosis. In 3 families, we identified a missense (p.Thr325Ser) and a nonsense
(p.Tyr429*) mutation in SEPSECS, encoding the O-phosphoseryl-tRNA:selenocysteinyl-tRNA
synthase, which was previously associated with progressive cerebellocerebral atrophy. We show
that the mutations do not completely abolish the activity of SEPSECS, but lead to decreased
selenoprotein levels, with demonstrated increase in oxidative protein damage in the patient brain.
Conclusions: These results extend the phenotypes caused by defective selenocysteine biosynthe-
sis, and suggest SEPSECS as a candidate gene for progressive encephalopathies with lactate
elevation. Neurology
®
2015;85:1–10
GLOSSARY
PCH2D 5pontocerebellar hypoplasia type 2D; PEHO 5progressive encephalopathy with edema, hypsarrhythmia, and optic
atrophy; RC 5respiratory chain; SRM-MS 5selected reaction monitoring–mass spectrometry; T
4
5thyroxine; tRNA 5
transfer RNA; TSH 5thyroid-stimulating hormone; T
3
5triiodothyronine.
Mitochondrial dysfunction is a frequent cause of childhood encephalopathy. Besides the typical
multisystemic disorders, an increasing number of mitochondrial defects are shown to cause a
CNS-specific phenotype.
1–5
Lactate elevation raises suspicion of mitochondrial involvement
and may be observed even in encephalopathies in which muscle biopsies show normal mito-
chondrial respiratory chain (RC) function.
1–3,6
Within our cohort of pediatric patients, we
identified patients with an undefined cause of cerebellocerebral atrophy, seizures, severe spas-
ticity, and axonal neuropathy with lactate elevation. We report that despite many of the clinical
and neuropathologic signs pointing toward mitochondrial impairment, the patients had
novel mutations in the SEPSECS gene, which functions in cytoplasmic transfer RNA
(tRNA)-charging in the selenoprotein biosynthesis pathway. We describe the uniform clinical,
neuroradiologic, and neuropathologic features of this entity and a detailed mutation
*These authors contributed equally to this work.
From the Department of Medical Genetics, Haartman Institute (A.-K.A., H.T.), Folkhälsan Institute of Genetics and Neuroscience Center (A.-K.A.,
A.L., A.-E.L.), Research Programs Unit, Molecular Neurology, Biomedicum Helsinki (T.H., P.I., A.L., E.Y., A.-E.L.), University of Helsinki;
Departments of Clinical Genetics (A.-K.A.) and Neurology (A.S.), Helsinki University Central Hospital; Department of Pediatric Neurology
(T. Linnankivi, P.I., T. Lönnqvist, H.P.), Children’s Hospital, University of Helsinki and Helsinki University Central Hospital, Finland;
Department of Biochemistry and Molecular Genetics (R.L.F., M. Simonovic), University of Illinois at Chicago; Department of Molecular Bio-
physics and Biochemistry (Y.L., D.S.), Yale University, New Haven, CT; Norio Centre (M. Somer), Department of Medical Genetics, Helsinki,
Finland; Turku Centre for Biotechnology (D.M.-P., G.L.C.), University of Turku and Åbo Akademi University; Department of Pediatric Neu-
rology (M.L.), South Karelia Central Hospital, Lappeenranta; Department of Radiology (L.V.), HUS Medical Imaging Center, Helsinki; and
Department of Pathology (A.P.), HUSLAB and University of Helsinki, Finland. G.L.C. is currently affiliated with Van’t Hoff Institute for
Molecular Sciences, University of Amsterdam, the Netherlands.
Go to Neurology.org for full disclosures. Funding information and disclosures deemed relevant by the authors, if any, are provided at the end of the article.
© 2015 American Academy of Neurology 1
ª2015 American Academy of Neurology. Unauthorized reproduction of this article is prohibited.
Published Ahead of Print on June 26, 2015 as 10.1212/WNL.0000000000001787
characterization. Moreover, our results indi-
cate oxidative damage in the brain as part of
the pathogenic mechanism resulting from se-
lenoprotein deficiency.
METHODS Standard protocol approvals, registrations,
and patient consents. All patient and control samples were
taken according to the Declaration of Helsinki, with informed
consent. The study was approved by the review board of the
Helsinki University Central Hospital.
The patients were identified within a cohort of 64 clinically
similar patients. One patient (patient 3) was part of the original
PEHO (progressive encephalopathy with edema, hypsarrhyth-
mia, and optic atrophy) syndrome patient series
7
and was
included in the neuroradiologic (group A) and ophthalmologic
study (patient 11) of that series.
A detailed neuropathologic examination was available for 3
patients (patients 1, 2, and 3), including the spinal cord from
patient 1; from patient 4, records pertaining to cerebellum, brain-
stem, and cerebral hemispheres were available. General autopsy
records were available from 3 patients (patients 1, 2, and 4).
Fresh-frozen tissue samples of patient 3 were available for the
study as well as fibroblasts of patients 1 and 2 and myoblasts of
patient 2.
DNA sequencing. For whole-exome sequencing, the exome
targets of the patients’DNA were captured with the
NimbleGen Sequence Capture 2.1M Human Exome v2.0 array
(NimbleGen, Basel, Switzerland) followed by sequencing with
the Illumina Genome Analyzer-IIx platform (Illumina, Inc.,
San Diego, CA) with 2 382 base pair paired-end reads. The
variant calling pipeline of the Finnish Institute for Molecular
Medicine was used for the reference genome alignment and
variant calling.
8
The coding exons of SEPSECS were sequenced
by Sanger sequencing.
Structural analysis of the mutations. Structural analysis was
based on the crystal structure of the human SEPSECS-tRNA
Sec
binary complex (PDB ID: 3HL2). The SEPSECS mutants
p.Thr325Ser and p.Tyr429*were generated in silico and analyzed
in Coot.
9
All figures were produced in PyMOL (The PyMOL
Molecular Graphics System, version 1.5.0.4, Schrödinger, LLC).
Oxyblot. The brain protein lysates were extracted using RIPA
buffer, and Oxyblot method was performed using an OxyBlot
Protein Oxidation Detection Kit (Millipore Corp., Billerica,
MA) according to the manufacturer’s instructions. The e-Meth-
ods on the Neurology
®
Web site at Neurology.org include full
descriptions of haplotype analysis, in vivo activity assay, and
protein analysis methods.
RESULTS Clinical data. We investigated 4 children
from 3 unrelated Finnish families. Clinical features
of the patients are summarized in table 1. These chil-
dren were born after uncomplicated pregnancies at
term to healthy nonconsanguineous parents. Two
patients were microcephalic at birth. The children
were irritable from birth and presented by the age
of 1 to 2 months with opisthotonus posturing, absent
head control, tremors, and myoclonic jerks. Severe
spastic or dystonic quadriplegia with absent psycho-
motor development became evident during the first
few months. Three patients had epileptic seizures,
including infantile spasms. As a sign of peripheral
neuropathy, the deep tendon reflexes attenuated or
vanished by the age of 2.5 years. The optic discs were
pale but not atrophic. All patients had edema of
hands, feet, and face, as well as narrow forehead,
tapering fingers, and high palate.
In 2 patients, early EEG studies (younger than 6
months of age) were normal, and later, hypsarrhythmia
with infantile spasms was documented. Later EEG re-
cordings showed severe slowing of background activity.
Sensory axonal neuropathy was verified by sural nerve
biopsy and electroneuromyography (table 1).
Two patients had elevated blood lactate levels and
one of them also had elevated lactate in the CSF. Pa-
tients 1 and 2 showed mild elevation of thyroid-
stimulating hormone (TSH) with normal levels of
thyroxine (T
4
) (table 1). Triiodothyronine (T
3
) levels
were not available. Other laboratory evaluations,
including liver transaminases, were unremarkable.
Neuroradiology and neuropathology showed neuron and
myelin loss. Neuroradiologic examinations showed
progressive cerebellar atrophy and less pronounced
cerebral atrophy (table e-1). Myelination was delayed
in the early MRIs and subsequently arrested. Cerebral
white matter showed pronounced and progressive
volume loss (figure 1).
Neuropathologic analysis revealed that all 4 pa-
tients shared features of progressive neuronal degener-
ation: laminar subtotal necrosis of the neocortex,
which was especially pronounced in the parietooccip-
ital regions (figure 2A), with relative sparing of the
hippocampi. The white matter showed myelin loss
and pallor with gliosis, consistent with degeneration
secondary to the neuronal loss. Patient 1 had subtotal
striatal degeneration (figure 2B) and also some tha-
lamic atrophy. All cases shared a quite severe degen-
eration and atrophy of the brainstem and cerebellar
cortex, creating an olivopontocerebellar atrophy–like
appearance: basis pontis, inferior olives, and tegmen-
tum of the medulla oblongata were severely atrophic;
the cerebellar cortex was severely atrophic as well
(figure 2, C and D). Here, the molecular layer was
thin, accompanied by a subtotal loss of Purkinje cells
and a very thin granule cell layer (figure 2E). The
spinal cord, available from one case, showed atrophy
and degeneration especially in the posterior columns.
Finally, the general autopsy revealed mild to moder-
ate mostly microvacuolar fatty degeneration of the
liver parenchyma (figure 2F).
SEPSECS mutations identified as the genetic cause. The
genetic cause of the disease was identified by whole-
exome sequencing of DNA samples of 2 patients
(patients 1 and 2) from 2 unrelated families. The
identified variants were first filtered to exclude
nongenic variants and those common in populations.
2Neurology 85 July 28, 2015
ª2015 American Academy of Neurology. Unauthorized reproduction of this article is prohibited.
On the basis that both families were of Finnish
ancestry and the clinical manifestation of both
patients closely resembled each other, we searched for
homozygous or compound heterozygous variants that
the patients shared. Two novel heterozygous variants
in SEPSECS were subsequently identified in both
patients, c.974C.G in exon 8 leading to a missense
mutation p.Thr325Ser and c.1287C.Ainexon11
leading to a nonsense mutation p.Tyr429*(RefSeq
NM_016955.3). The variants were validated by
Sanger sequencing (figure 3A). The parents of patient
1 and the mother of patient 2 were heterozygous
carriers; the DNA sample of the father of patient 2
was not available. The variants were not present in the
1000 Genomes database (www.1000genomes.org) or in
the NHLBI GO ESP Exome Variant Server (evs.gs.
washington.edu/EVS/) or in approximately 230
screened Finnish control samples. However, a recent
database of 3,323 exome sequences of Finnish
individuals provided a heterozygous carrier frequency
of 1:277 for the c.1287C.A, p.Tyr429*variant
(Sequencing Initiative Suomi, sisu.fimm.fi), suggesting
Table 1 Summary of clinical and laboratory examinations of patients
Study
Patient 1, family 1 Patient 2, family 2 Patient 3, family 3 Patient 4, family 3
Sex MFFF
Gestational age, wk 41 16 39 41 At term
Weight at birth, g 4,000 3,700 3,120 2,820
OFC at birth, cm (SD) 36.5 (11) 35.5 (10.5) 31 (23.3) 32 (22.3)
Presenting signs (age at
onset)
Irritability (neonatal),
opisthotonus (2 mo) Irritability, opisthotonus
(neonatal) Irritability, opisthotonus
(1.5 mo) NA
Tremors/myoclonus 111NA
Epileptic seizures (age at
onset)
Infantile spasms (12 mo) Infantile spasms (11 mo) 1NA
Spastic or dystonic
quadriplegia
1111
Central visual impairment 1111
Optic atrophy 222NA
Intellectual disability Profound Profound Profound Profound
Dysmorphic features
High arched palate 111NA
Tented upper lip 211NA
Outward turning ear
lobules
112NA
Narrow forehead 111NA
Edema of hands and feet 111NA
Tapered fingers 111NA
EEG (age) N (3 and 5 mo), hypsarrhythmia
(12 mo), very slow background
(6 y)
N (3 and 4 mo),
hypsarrhythmia (13 mo) Slow background (4 mo),
multifocal spikes, very slow
background (10 y)
Unspecific slowing
(14 mo)
Muscle biopsy N Variable fiber size,
neurogenic damage NNA
Sural nerve biopsy (age at
investigation)
Axonal neuropathy (2 y) Axonal neuropathy (2.3 y) Axonal neuropathy (10 y) NA
Neurography MCV low N, motor ampl N,
sensory ampl: radialis Y, suralis
—(6 y)
MCV low N, Motor ampl N,
Sensory ampl: radialis Y,
suralis—(2.3 y)
NA NA
Blood lactate (ref. 0.7–1.8
mmol/L)
4.9, 0.8, 5.6, 3.1 0.4, 2.8, 5.9, 4.2 N NA
CSF lactate (ref. 0.6–2.7
mmol/L)
1.5 1.4, 4.2 1.7 NA
Age at death, y 8.5 4.3 15.3 2.7
Abbreviations: ampl 5amplitudes; MCV 5motor nerve conduction velocity; N 5normal; NA 5not available; OFC 5occipital frontal circumference; ref. 5
reference.
Symbols: 25absent; 15present; Y5decreased.
Neurology 85 July 28, 2015 3
ª2015 American Academy of Neurology. Unauthorized reproduction of this article is prohibited.
enrichment of the nonsense variant in Finland, but
without homozygous occurrence.
Next we screened 11 SEPSECS exons for mutations
in additional patients with mitochondrial encephalopa-
thy and/or other shared features. One patient (patient 3)
from an affected sib-pair with similar clinical findings
was found to be compound heterozygous for the
same SEPSECS mutations c.974C.G(p.Thr325Ser)
and c.1287C.A(p.Tyr429*). The DNA sample of
the affected sib was not available for the study. Inves-
tigation of the family histories of the 3 patients with
shared SEPSECS mutations revealed that they all
originated from a restricted area in eastern Finland.
Haplotype analysis of the nearby microsatellite markers
indicated shared ancestral haplotypes, further support-
ing the distant common origin of the mutations in our
patients (figure 3B).
Predicted effects of the mutations onto the SEPSECS
protein structure. SEPSECS codes for O-phosphoseryl-
tRNA:selenocysteinyl-tRNA synthase, the key en-
zyme in the sole biosynthetic route to selenocysteine
(Sec) in eukaryotes and archaea.
10,11
Residue Thr325,
which is affected by a missense mutation in our patients,
is a highly conserved amino acid in eukaryotic
SEPSECS (figure 3C). In the SEPSECS structure,
Thr325 is located in the middle of helix a12 in the
C-terminal domain of the protein (figure 3D). Helix
a12 provides support for the major elements that con-
stitute the active site of SEPSECS. Thr325 interacts
only with the backbone and side-chain atoms of helix
a12, and its replacement with serine may destabilize the
structure of a-helix. This could lead to altered
positioning of the cofactor pyridoxal phosphate, and
the floor (helix a10) and the clefts (loop 70 and
P-loop) of the active site. These structural
rearrangements in the catalytic pocket would
ultimately yield an enzyme with reduced catalytic
power.
The p.Tyr429*mutant messenger RNA escapes
nonsense-mediated decay, and is predicted to lead to
truncation of the 73 C-terminal amino acids of SEP-
SECS, the region critical both for tRNA binding and
enzyme activity (figure e-1). The full-length SEP-
SECS of approximately 55 kDa was readily detected
by Western blotting—even in a higher amount than
in the control sample (figure 3E)—in the brain
autopsy sample of patient 3, but no evidence of a
truncated protein (;47 kDa) was found, indicating
that it was either not produced or rapidly degraded.
Figure 1 Brain imaging findings of SEPSECS deficiency
T2 and fluid-attenuated inversion recovery images of patient 1 at age 5 months (A) and 2 years, 10 months (B, C) and patient
2 at age 6 months (D) and 1 year, 8 months (E, F). The early images (A, D) show loss of periventricular white matter volume
and almost complete lack of myelin (arrows). Upon disease progression (B, C, E, F), widening of both central and cortical CSF
spaces and cerebellar atrophy are present, with no signal for myelin.
4Neurology 85 July 28, 2015
ª2015 American Academy of Neurology. Unauthorized reproduction of this article is prohibited.
To functionally confirm the mutation effects, we
showed that both mutations severely affected
SEPSECSactivityinvivoinananaerobicEsche-
richia coli assay.
12
For the assay, we utilized the
DselA strain and inspected the ability of human
SEPSECStorestorethebenzylviologen–reducing
activity of an Ecoliselenoprotein, the formate
dehydrogenase H. As predicted, p.Tyr429*mutant
was completely inactive in this assay, while
the p.Thr325Ser mutant was active albeit at the
decreased level (figure 3F).
Functional consequences of the SEPSECS mutations. In-
activating SEPSECS mutations presumably inhibit
synthesis of 25 selenoproteins (the human selenopro-
teome), which participate in diverse biological processes.
13
We measured selenoprotein levels in the lysate ob-
tained from the autopsy brain material of patient 3
using selected reaction monitoring–mass spectro-
metry (SRM-MS). Protein levels were normalized
against glyceraldehyde 3-phosphate dehydrogenase
andphosphoglyceratekinase1.Tovalidatethe
method, the levels of the glial fibrillary acidic protein,
Figure 2 Histologic findings of SEPSECS deficiency
(A) Parietooccipital cortex displaying edemic transcortical laminar necrosis (ln) with a subtotal neuronal loss (arrows). (B)
Putamen (put) is severely neuron-depleted and gliotic (arrows). (C) Pontine basis (pb) is narrow, both neurons and transverse
fibers are reduced. (D) Atrophy of the inferior olives (io) and hili with olivocerebellar fibers and narrowed tegmentum seen in
low magnification of the medulla oblongata. (E) Atrophic cerebellar cortex, the molecular and granular layers are thin, with
practically total loss of Purkinje cells (arrows). (F) Liver parenchyma exhibiting a moderate microvacuolar fatty degeneration
(fat visible as white droplets). Paraffin sections, hematoxylin & eosin (A, C, D, F), Luxol fast blue–cresyl violet (B, E); original
magnification 340 (A, E), 3100 (B), 310 (C, D), 3400 (F).
Neurology 85 July 28, 2015 5
ª2015 American Academy of Neurology. Unauthorized reproduction of this article is prohibited.
which resides in astrocytes, were measured and shown
to be increased, thus indicating astrogliosis (figure 4,
A, D). In contrast, levels of myelin basic protein
and neurofilament medium were clearly reduced
(figure 4A), which is consistent with the observed
myelin and neuron loss in the patients. Three
selenoproteins, thioredoxin reductase TXNRD1 and
glutathione peroxidases GPX1 and GPX4, were
Figure 3 SEPSECS gene defect with structural and functional consequences of the mutations
(A) SEPSECS mutation sequences. The arrow indicates th em utation site. (B) Haplotypes on the chromosome 4 region containing
the SEPSECS gene in 3 patients from 3 unrelated families. Parent samples were used to construct the haplotypes when
available. (C) Alignment of the sequence region containing Thr325 (ar row) in eukaryo tic SEPSECS pr oteins. (D) Thr 325 is located
in the middle of helix a12 in the C-terminal domain of the protein. Helix a12 provides support for the major elements that
constitute the active site of SEPSECS by 2 direct interactions: (1) helix a12 (orange) interacts with helix a10 (from monomer 1 ,
in gray); (2) helix a10 carries a catalytic residue Lys284 (orange sticks) to which the cofactor PLP (orange sticks) is covalently
attached and forms the floor of the active site. The a10–a12 interaction places Thr325 (orange sticks) behind the active-site
pocket and approximately 15 Å away from PLP (shown with up-and-down arrow). In addition, helix a12 interacts with helix a3
(blue) from the second SEPSECS monomer (monomer 2, in light blue), which is flanked by loop 70 and P-loop (blue) that form the
cleftsoftheactivesite.TheCCAendoftRNA
Sec
(beige) binds in the proximity of the P-loop, which is also implicated in binding of
the phosphoseryl group. (E) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis from brain samples; SEPSECS patient
(P) and control (C). The full-length SEPSECS of approximately 55 kDa was readily detected, but no signal of the size of the
truncated protein (;47 kDa)was present.(F) In vivo dilution series assay of the ability of humanSEPSECS variants to restore the
benzyl viologen–reducing activity of the selenoprotein formate dehydrogenase H in the Escherichia coli DselA deletion strain.
GAPDH 5glyceraldehyde 3-phosphate dehydrogenase; PLP 5pyridoxal phosphate; tRNA 5transfer RNA.
6Neurology 85 July 28, 2015
ª2015 American Academy of Neurology. Unauthorized reproduction of this article is prohibited.
abundant enough to be reliably detected. Their levels
were decreased by 15% to 40% in the patient brain
sample compared with controls (figure 4B). Consistent
with a partial defect in selenoprotein production, levels
of TXNRD1 and TXNRD2 were decreased in the
patient’s brain as shown by Western blotting (figure
4D). However, the steady-state level of tRNA
Sec
was
not altered (figure 4F). Of note, the observed defects
were tissue-specific, as the patient’s fibroblasts,
myoblasts, or differentiated myotubes did not show
reduced TXNRD levels (data not shown).
Because of the lactate elevation, we analyzed the
amounts of mitochondrial RC complexes I–IV in
the autopsy brain samples of patient 3 by blue native
electrophoresis, but found them to be similar to con-
trols (figure 4E). In addition, when we quantified the
amounts of RC complex subunits by SRM-MS, they
were shown to be mostly unaffected (figure 4C).
Figure 4 Selenoprotein and respiratory chain protein amounts in SEPSECS deficiency
(A–C) Selected reaction monitoring–mass spectrometry; autopsy brain sample of patient 3 and 3 controls. The results are
shown for (A) glial fibrillary acidic protein (GFAP), myelin basic protein (MBP), and neurofilament medium (NFM), markers of
brain cells, for (B) selenoproteins glutathione peroxidases 1 and 4 (GPX1 and GPX4) and thioredoxin reductase 1 (TRXR1)
and for (C) mitochondrial respiratory chain complex subunits: NADH dehydrogenase (ubiquinone) Fe-S protein 1 (NDUFS1),
NADH dehydrogenase (ubiquinone) Fe-S protein 8 (NDUFS8), ubiquinol-cytochrome creductase core protein II (QCR2),
cytochrome coxidase subunit Va (COX5A), mitochondrially encoded ATP synthase 6 (ATPA), and citrate synthase (CISY).
(D) Western blotting of the brain samples for GFAP and 2 selenoproteins (P, patient 3; C, controls 1–3). (E) Blue native
electrophoresis of mitochondrial respiratory chain complexes in the patient (P) and control (C) brain samples. (F) Steady-
state levels of tRNA
Sec
compared with mitochondrial tRNA
Ala
in the brain sample of patient 3 (P) and controls (C1–C4). (G)
Oxyblot shows increased amounts of oxidized proteins in patient brain (P) compared with controls (C1–C3). GAPDH 5
glyceraldehyde 3-phosphate dehydrogenase; mt-tRNA 5mitochondrial transfer RNA; tRNA
Sec
5selenocysteine-specific
transfer RNA.
Neurology 85 July 28, 2015 7
ª2015 American Academy of Neurology. Unauthorized reproduction of this article is prohibited.
Because the decreased selenoprotein synthesis
affected glutathione peroxidases and thioredoxin
reductases—enzymes of antioxidant defense—we
analyzed protein carbonylation in the patient’s
brain, and found protein oxidation to be clearly
increased (figure 4G).
DISCUSSION We report here that pathogenic muta-
tions in SEPSECS lead to severe cerebellocerebral
atrophy by attenuating the synthesis of selenopro-
teins, which leads to considerable oxidative damage
in the patient brains.
SEPSECS mutations were previously described in
a single report, underlying progressive cerebellocere-
bral atrophy with profound mental retardation, pro-
gressive microcephaly, severe spasticity, and
myoclonic or generalized tonic-clonic seizures, later
classified as pontocerebellar hypoplasia type 2D
(PCH2D) (MIM 613811).
12,14
The MRI findings
of progressive cerebellar atrophy, followed by cerebral
atrophy involving both white and gray matter, mim-
icked the findings in our patients. In contrast to a
normal metabolic profile of patients with PCH2D,
12
our patients had lactacidemia, and they also presented
with axonal neuropathy. Similarities between the pa-
tients with PCH2D and the patients described here
support the disease-causing role of the identified mu-
tations, thus extending the phenotypes caused by
defective selenocysteine biosynthesis.
The neuropathologic changes of SEPSECS defi-
ciency, such as progressive neuronal degeneration,
more pronounced laterally than midline, are remi-
niscent of Alpers syndrome,
15
which is caused by
defects in mitochondrial proteins, most commonly
in polymerase gamma, but also in Twinkle helicase
and the phenylalanyl-tRNA synthetase.
4,16,17
The
topography of the lesions corresponds to highly
energy-dependent regions of the CNS; in case of
SEPSECS deficiency, this is particularly the parieto-
occipital region. The patients with SEPSECS muta-
tions described here also showed moderate
degeneration of the liver postmortem. These find-
ings, together with elevated lactate, suggest that
encephalopathy due to SEPSECS deficiency and
mitochondrial encephalopathies, such as Alpers syn-
drome, could share some common pathogenic
mechanisms. However, we did not identify signifi-
cant alterations in the mitochondrial RC complexes
in the patient’s brain sample that would explain
changes typical for RC deficiencies.
Similar manifestations between mitochondrial and
selenoprotein disorders could be explained by the role
selenoproteins have in maintaining the cellular redox
potential and H
2
O
2
detoxification.
13
In other words,
selenoproteins are an important part of the antioxi-
dant defense. We showed remarkable increase of
protein carbonylation as a sign of oxidative stress in
the brain of patients with SEPSECS deficiency. As
mitochondria are one of the main sources of cellular
reactive oxygen species, SEPSECS deficiency could
especially damage cells with high mitochondrial activ-
ity. Future work on mouse models is needed to clarify
this aspect of selenoprotein pathology.
Progressive cerebellar atrophy of our patients
together with infantile spasms, dysmorphic features,
and edema of hands, feet, and face resembled the
findings typically seen in PEHO syndrome.
7,18
How-
ever, unlike patients with PEHO, our patients did
not have optic atrophy and were spastic rather than
hypotonic. Previously, a connection between ponto-
cerebellar hypoplasias, mitochondrial encephalopa-
thies, and PEHO-like features has been proposed in
PCH6 that is caused by RARS2 mutations.
19
RARS2
encodes the mitochondrial aminoacyl-tRNA synthe-
tase essential for charging tRNA
Arg
for protein synthe-
sis of mitochondrial RC complexes.
6
Of note,
SEPSECS also affects cellular functions through
tRNA, but in a different biological pathway, by cat-
alyzing the conversion of the phosphoseryl-tRNA
Sec
intermediate into selenocysteinyl-tRNA
Sec
.
10,11
Fur-
thermore, PCH2 types A, B, and C are caused by
mutations in genes encoding for subunits of the
tRNA-splicing endonuclease complex, TSEN54,
TSEN2 and TSEN34, respectively.
20
This complex
performs the splicing of intron-containing tRNAs,
which comprise approximately 6% of human tRNAs
needed for cytoplasmic translation.
21
Whether a com-
mon mechanism exists by which the defects of the
different cellular protein synthesis processes lead to
similar neurodegenerative phenotypes or whether
tRNA involvement in all these entities is purely coin-
cidental remains to be established.
SEPSECS is a tetramer composed of 2 dimers,
where each dimer contains 2 active sites formed at
the dimer interface.
10,11
The novel compound heter-
ozygous mutations identified in this study were pre-
dicted to have differential effects on the function of
the tetrameric SEPSECS: p.Thr325Ser introduced
Ser in the middle of an a-helix predicting destabili-
zation of the helix,
22
thereby modifying the catalytic
pocket of the enzyme, whereas p.Tyr429*resulted in
a total loss of function. Our dilution series of the
bacterial in vivo assay verified these predictions.
The previously reported SEPSECS mutations of pa-
tients with PCH2D displayed no detectable activity
in an anaerobic in vivo assay for SEPSECS activity.
12
Thus, the differences in the phenotypes of our pa-
tients and those reported previously may be caused by
differences in the residual SEPSECS activity. The fact
that the fibroblasts, myoblasts, and myotubes of our
patients did not display decreased selenoprotein levels
(not shown) suggests that the residual SEPSECS
8Neurology 85 July 28, 2015
ª2015 American Academy of Neurology. Unauthorized reproduction of this article is prohibited.
activity was sufficient to maintain selenoprotein syn-
thesis in these cell types but not in the brain.
The 25 human selenoproteins function in remark-
ably diverse processes. Besides SEPSECS deficiency,
the only known human disease affecting selenopro-
tein synthesis is caused by mutations in SECISBP2,
which encodes a protein that recognizes the specific
insertion sequence in the selenoprotein messenger
RNAs. SECISBP2 mutations cause either a multisys-
tem disorder
23
or abnormal thyroid hormone metab-
olism with elevated TSH, T
4
, and reverse T
3
and
reduced T
3
levels.
24
Our patients presented with
increased TSH and normal T
4
levels, as measured
at ages 2 and 6 years. Thus, although affecting the
common metabolic pathway, mutations in SE-
CISBP2 and SEPSECS yield distinct phenotypes.
The selenoproteome is essential for mammals as
shown by the early embryonic lethality of the tRNA
Sec
gene (Trsp) knockout mouse.
25
The selenoprotein
knockout mice have further clarified their importance
for brain function.
26
The neuron-specific knockout
mice of GPx4,
27
Txnrd1,
28
and Trsp
29
are characterized
by neurodegeneration, in general, and by cerebellar
hypoplasia, in particular.
30
In the Txnrd1 mice, the
effect was attributed to decreased proliferation of gran-
ule cell precursors within the external granular layer.
28
Furthermore, the full knockout of SelP,
31
which enco-
des a plasma protein SELP that transports selenium
from the liver to peripheral tissues, was shown to have
a neurologic phenotype with spasticity and seizures.
32
It
is likely that the lack of several selenoproteins contrib-
utes to the neuronal loss in the selenoprotein biosyn-
thesis defects. Our SRM-MS and Western blotting
results derived from the analysis of an autopsy brain
sample from a patient with SEPSECS mutations
showed that selenoproteins were present albeit at sig-
nificantly reduced levels. However, because of the
severe neuronal loss and astrogliosis in the patient brain,
a comparative analysis has limitations. For instance,
neurons may have had more severe reductions of sele-
noproteins before their death. Also, the measurement of
selenoenzyme activities was not feasible with this mate-
rial. Regardless of these shortcomings, our result of
SEPSECS mutations causing a selenoprotein deficiency
in human brain implicates a specific requirement of
selenoproteins in postnatal brain development.
Based on the results of our study, we suggest SEP-
SECS sequencing in progressive early childhood brain
atrophies of unknown cause, especially when patients
present with sensory axonal neuropathy and elevated
lactate.
AUTHOR CONTRIBUTIONS
A.-K.A. collected and analyzed patient data and drafted the manuscript.
T.H., A.L., and E.Y. sequenced patients and performed protein and cell
culture experiments. T. Linnankivi and P.I. contributed to the clinical
analysis. R.L.F. and M. Simonovic performed the structural analysis.
Y.L. and D.S. performed the in vivo bacterial assay. D.M.-P. and
G.L.C. performed the mass spectrometry analysis. M. Somer, M.L.,
T. Lönnqvist, and H.P. contributed to the clinical analysis. L.V. per-
formed neuroradiology. A.P. performed neuropathology. H.T. performed
the exome sequencing analysis. A.-E.L., A.S., and H.T. designed the
study and drafted the manuscript. All authors revised the manuscript.
ACKNOWLEDGMENT
Anu Harju and Riitta Lehtinen are thanked for technical help. The authors
acknowledge the exome capture, sequencing, and variant calling pipeline
analysis performed by the Institute for Molecular Medicine Finland
FIMM, Technology Centre, and University of Helsinki. Proteome analysis
was made possible through use of the National Proteomics and Metabolo-
mics infrastructure of Biocenter Finland.
STUDY FUNDING
The authors thank the Sigrid Jusélius Foundation, Academy of Finland
and University of Helsinki (to H.T. and A.S.), Jane and Aatos Erkko
Foundation (to A.S.), Folkhälsan Research Foundation (to A.-E.L.), the
US NIH grant GM097042 (to M. Simonovic), the US National Institute
of General Medical Sciences GM22854 (to D.S.), Arvo and Lea Ylppö
Foundation (to A.-K.A. and H.T.), Orion Farmos Research Foundation
(to H.T.), Helsinki University Central Hospital Research Fund (to A.-K.
A.), and Foundation for Pediatric Research (to P.I.) for funding support.
DISCLOSURE
A. Anttonen has received research support from Arvo and Lea Ylppö
Foundation and Helsinki University Central Hospital Research Fund.
T. Hilander and T. Linnankivi report no disclosures relevant to the
manuscript. P. Isohanni has received research support from Foundation
for Pediatric Research. R. French and Y. Liu report no disclosures rele-
vant to the manuscript. M. Simonovic has received research support from
the US NIH grant GM097042. D. Söll received research support from
the US National Institute of General Medical Sciences (GM22854).
M. Somer, D. Muth-Pawlak, G. Corthals, A. Laari, E. Ylikallio,
M. Lähde, L. Valanne, T. Lönnqvist, H. Pihko, and A. Paetau report
no disclosures relevant to the manuscript. A. Lehesjoki has received
research support from the Folkhälsan Research Foundation. A. Suoma-
lainen has received research support from Sigrid Jusélius Foundation,
Academy of Finland, University of Helsinki, and Jane and Aatos Erkko
Foundation. H. Tyynismaa has received research support from Arvo and
Lea Ylppö Foundation, Orion Farmos Research Foundation, Sigrid
Jusélius Foundation, Academy of Finland, and University of Helsinki.
Go to Neurology.org for full disclosures.
Received November 17, 2014. Accepted in final form March 26, 2015.
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10 Neurology 85 July 28, 2015
ª2015 American Academy of Neurology. Unauthorized reproduction of this article is prohibited.
DOI 10.1212/WNL.0000000000001787
published online June 26, 2015Neurology
Anna-Kaisa Anttonen, Taru Hilander, Tarja Linnankivi, et al.
lactate
Selenoprotein biosynthesis defect causes progressive encephalopathy with elevated
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