Recessive loss of function of the neuronal ubiquitin hydrolase UCHL1 leads to early-onset progressive neurodegeneration
Ubiquitin C-terminal hydrolase-L1 (UCHL1), a neuron-specific de-ubiquitinating enzyme, is one of the most abundant proteins in the brain. We describe three siblings from a consanguineous union with a previously unreported early-onset progressive neurodegenerative syndrome featuring childhood onset blindness, cerebellar ataxia, nystagmus, dorsal column dysfuction, and spasticity with upper motor neuron dysfunction. Through homozygosity mapping of the affected individuals followed by whole-exome sequencing of the index case, we identified a previously undescribed homozygous missense mutation within the ubiquitin binding domain of UCHL1 (UCHL1(GLU7ALA)), shared by all affected subjects. As demonstrated by isothermal titration calorimetry, purified UCHL1(GLU7ALA), compared with WT, exhibited at least sevenfold reduced affinity for ubiquitin. In vitro, the mutation led to a near complete loss of UCHL1 hydrolase activity. The GLU7ALA variant is predicted to interfere with the substrate binding by restricting the proper positioning of the substrate for tunneling underneath the cross-over loop spanning the catalytic cleft of UCHL1. This interference with substrate binding, combined with near complete loss of hydrolase activity, resulted in a >100-fold reduction in the efficiency of UCHL1(GLU7ALA) relative to WT. These findings demonstrate a broad requirement of UCHL1 in the maintenance of the nervous system.
Recessive loss of function of the neuronal ubiquitin
hydrolase UCHL1 leads to early-onset
, Navneet K. Tyagi
, Cigdem Ozkara
, Beyhan Tuysuz
, Mehmet Bakircioglu
, Murim Choi
, Ahmet O. Caglayan
, Jacob F. Baranoski
, Ozdem Erturk
, Cengiz Yalcinkaya
, Murat Karacorlu
, Michele H. Johnson
, Shrikant Mane
, Sreeganga S. Chandra
, Angeliki Louvi
, Titus J. Boggon
Richard P. Lifton
, Arthur L. Horwich
, and Murat Gunel
Genetics, Program on Neurogenetics,
Molecular, Cellular, and
Developmental Biology, and
Howard Hughes Medical Institute, Yale School of Medicine, New Haven, CT 06510;
Division of Genetics, Department of Pediatrics, Istanbul University Cerrahpasa Faculty of Medicine, Istanbul 34098, Turkey;
Institute, Istanbul 34349, Turkey; and
Department of Radiology, Acibadem University School of Medicine, Istanbul 34742, Turkey
Contributed by Arthur L. Horwich, December 31, 2012 (sent for review November 17, 2012)
Ubiquitin C-terminal hydrolase-L1 (UCHL1), a neuron-speciﬁc de-
ubiquitinating enzyme, is one of the most abundant proteins in
the brain. We describe three siblings from a consanguineous union
with a previously unreported early-onset progressive neurode-
generative syndrome featuring childhood onset blindness, cere-
bellar ataxia, nystagmus, dorsal column dysfuction, and spasticity
with upper motor neuron dysfunction. Through homozygosity
mapping of the affected individuals followed by whole-exome se-
quencing of the index case, we identiﬁed a previously undescribed
homozygous missense mutation within the ubiquitin binding
domain of UCHL1 (UCHL1
), shared by all affected subjects.
As demonstrated by isothermal titration calorimetry, puriﬁed
, compared with WT, exhibited at least sevenfold re-
duced afﬁnity for ubiquitin. In vitro, the mutation led to a near
complete loss of UCHL1 hydrolase activity. The GLU7ALA variant
is predicted to interfere with the substrate binding by restricting
the proper positioning of the substrate for tunneling underneath
the cross-over loop spanning the catalytic cleft of UCHL1. This in-
terference with substrate binding, combined with near complete
loss of hydrolase activity, resulted in a >100-fold reduction in the
efﬁciency of UCHL1
relative to WT. These ﬁndings demon-
strate a broad requirement of UCHL1 in the maintenance of the
protein quality control
recessive inherited neurodegeneration
Neurodegenerative syndromes represent a diverse group of
disorders characterized by progressive neurological decline,
typically associated with an anatomical correlate. Although the
clinical onset of most of the common neurodegenerative disorders,
such as Parkinson or Alzheimer’s diseases, is during adulthood (1),
some syndromes become manifested during childhood. Despite
being exceedingly rare in the population, understanding the mo-
lecular basis of these early-onset neurodegenerative syndromes
could allow a unique insight into mechanisms of central nervous
system maintenance. To gain a biological understanding of such
disorders, we focused on consanguineous kindreds in which the
affected subjects presented to medical attention during childhood
with neurological decline. In one such kindred, NG 1024 (Fig. 1A),
which originated from Turkey, three of the six siblings, who were
offspring of a ﬁrst-cousin consanguineous union, were found to
have vision loss during early childhood, followed by progressive
neurological dysfunction of the pyramidal system, cerebellum, and
spinal dorsal columns. To identify the genetic basis of this syn-
drome with apparent autosomal recessive mode of inheritance,
we initially used whole genome genotyping to map the homo-
zygous segments shared by affected family members, which
presumably contained the disease-causing mutation. We then
performed whole-exome capture and next-generation sequencing
of the index case. Application of next-generation sequencing tech-
nologies, speciﬁcally exome sequencing, which allows for selective
sequencing of all exons, has previously been proven to be the most
efﬁcient and cost-effective approach in the discovery of disease-
causing variants in various Mendelian disorders (2–4), such as this
family. Based on the results of the exome sequencing, we analyzed
each variant located within the homozygous regions shared by all
affected members. We identiﬁed a homozygous missense mutation
affecting a glutamic acid residue within the ubiquitin binding do-
main of the ubiquitin C-terminal hydrolase L1 (UCHL1)gene
) that cosegregated with the phenotype.
UCHL1 is one of the most abundant proteins in the brain,
comprising 1–2% of the total soluble fraction (5). It is a neuron-
speciﬁc de-ubiquitinating enzyme and plays an important role
in ubiquitin turnover through its C-terminal hydrolytic activity.
UCHL1 has also been suggested to be a ubiquitin ligase (6). Al-
though UCHL1 has previously been implicated in the patho-
physiology of neurodegenerative disorders including Parkinson
(7) and Alzheimer’s (8) diseases, conclusive evidence that links
UCHL1 dysfunction to neurodegeneration has been lacking.
Here, based on the molecular genetics data that led to the iden-
tiﬁcation of the UCHL1
mutation in the family under
study, followed by the molecular studies that demonstrated the
GLU7ALA mutation to nearly completely abolish UCHL1’s hy-
drolase activity, we conclude that compromised UCHL1 activity
leads to a childhood-onset multisystem neurodegenerative syn-
drome. Our ﬁndings link loss of UCHL1 function with broad
neurodegeneration and demonstrate its fundamental importance
in the maintenance of the nervous system.
Early-Onset Neurodegenerative Syndrome. All three affected chil-
dren were products of uncomplicated, term labors and reached early
neurodevelopmental milestones normally. They began suffering
from vision loss at around age 5 y, followed by slowly progressive
neurological problems. After three decades, all had developed
blindness, cerebellar ataxia with an inability to stand without
Author contributions: K.B., N.K.T., A.L.H., and M.G. designed research; K.B., N.K.T., C.O.,
B.T., M.B., S.D., A.O.C., J.F.B., O.E., C.Y., M.K., A.D., M.H.J., S.M., S.S.C., and A.L. performed
research; M.C. and R.P.L. contributed new reagents/analytic tools; K.B., N.K.T., S.S.C., A.L.,
T.J.B., A.L.H., and M.G. analyzed data; and K.B., N.K.T., A.L., R.P.L., A.L.H., and M.G. wrote
The authors declare no conﬂict of interest.
Freely available online through the PNAS open access option.
K.B. and N.K.T. contributed equally to this work.
To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
www.pnas.org/cgi/doi/10.1073/pnas.1222732110 PNAS Early Edition
Fig. 1. Identiﬁcation of the GLU7ALA (E7A) mutation in UCHL1 in kindred NG 1024. (A) Simpliﬁed pedigree is shown; affected subjects are denoted by ﬁlled
symbols (arrow indicates the index case). (B) Coronal MR images of NG 1024–3(Right) and a control subject (Left) reveal diffuse cerebral and optic chiasmal
atrophy (red arrowhead) in the index case. Cortical atrophy is indicated by cerebral volume loss with increase in the subarachnoid and intersulci spaces over
the brain, which are ﬁlled with cerebrospinal ﬂuid (black in these images). (C) Axial orbital MR images of a control subject (Left) and NG 1024–3(Right) show
optic nerve (red arrowhead) atrophy. (D) Sagittal MR images of a control subject (Left) and NG 1024–3(Right) reveal cerebral cortical, cerebellar (red circle),
and optic tract (red arrowheads) atrophy. (E) Exome sequencing reveals an A to C change. The WT sequence in blue is shown on top, with the mutant base in
red below. There was 35-fold coverage, with all reads revealing the substitution. (F) Sanger sequencing conﬁrms the mutation that results in glutamic acid
(E, in blue) to alanine (A, in red) change. The unaffected parents are heterozygous for the variant (Right).
www.pnas.org/cgi/doi/10.1073/pnas.1222732110 Bilguvar et al.
assistance, nystagmus, and titubation (head tremor) (Table S1;SI
Materials and Methods). Muscle strength was grossly normal in all
subjects, with the male subject suffering from myotonia (muscles
failing to relax after activity). There was decreased vibration and
position sense due to dorsal column dysfunction. Deep tendon
reﬂexes were increased throughout with spasticity, indicating upper
motor neuron dysfunction. There were no extrapyramidal signs.
Outside the nervous system, there was no apparent phenotype.
The parents and remaining three siblings were healthy.
Further laboratory tests conﬁrmed the ﬁndings on physical
examination (Figs. S1 and S2). Consistent with the clinical ﬁnding
of blindness, ﬂash visual-evoked potentials revealed nearly absent
response in both eyes but normal full-ﬁeld electroretinograms.
Nerve conduction velocities were normal, but myokymic activity
was detected in all muscles tested during EMG (Fig. S1). So-
matosensory-evoked potential (SSEP) studies suggested dorsal
column dysfunction that was more signiﬁcant in the lower ex-
tremities (Fig. S2).
In all three patients, MRI scans were remarkable for bilateral
optic nerve and chiasm atrophy, with diffusion tensor imaging
(DTI) studies revealing bilateral Wallerian degeneration of the
optic radiations (Fig. 1 B–D;Table S2) (9, 10). The MRI scans
were also remarkable for obvious cerebellar and mild cerebral
atrophy that was more severe in the older siblings (Fig. 1 Band D).
The ﬁnding of a rare phenotype recurring in a sibship from a con-
sanguineous union suggests autosomal recessive transmission.
Whole-Exome Sequencing Identiﬁed a Missense Variant, GLU7ALA, in
UCHL1.We initially performed whole-genome genotyping of the
three affected siblings using Illumina 610K SNP chips and con-
ﬁrmed the reported consanguinity. Slightly greater that what is to
be expected for a consanguineous ﬁrst-cousin union, inbreeding
coefﬁcients of the affected subjects ranged between 9.6% and
10.6%, and the number of the homozygous segments (>2.5 cM) in
each patient was between 23 and 34, with the total size of the
homozygous regions being between 241.3 and 358.7 million bp
(MB) (324.08 and 382.09 cM, respectively). Among these homo-
zygous segments, only four chromosomal regions, on chromo-
somes 4, 6, 8, and 11, were shared by all three affected siblings.
These regions, which together comprised 27.16 MB, presumably
contained the disease-causing mutation (Table S3). We next
performed whole-exome sequencing of the index case using
Nimblegen solid-phase array capture and the Illumina Genome
Analyzer IIx instrument and focused on variants located within
these shared regions (2–4, 11). A single lane of sequencing on
Illumina’s Genome Analyzer IIX with single-read chemistry and
a read length of 74 bases yielded ∼38 million reads. These data in
turn achieved a high and uniform coverage across the targeted
bases with a mean coverage of ∼52-fold. Nearly 99% and 97% of
all of the targeted bases within the homozygosity intervals were
read at least four and eight times, respectively. The mean se-
quence error rate was 0.97% (Table S4). The sensitivity and
speciﬁcity for the detection of homozygous variation from the
reference human genome were high (98.75% and 99.75%, re-
spectively) as determined by comparison of sequencing data to the
results of SNP genotyping as a reference.
Although a total of 141 homozygous variants were detected within
these homozygous segments, only ﬁve of these variants were not
previously identiﬁed in dbsnp (build 131) and 1,000 genomes project
databases and among 2,400 exomes of European subjects sequenced
at Yale (Table 1; Table S5), and two of the homozygous variants
altered the encoded protein. The ﬁrst of these variants, a proline to
leucine substitution in ZNF259(ZNF259
11: 116,658,654 G >A), failed to segregate with the disease phe-
notype—the father and one of the unaffected siblings were ho-
mozygous for the variant. In contrast, the second variant, an A to C
transversion on chromosome 4, position 41,259,013, cosegregated
with the trait and was absent both from the Yale cohort and 948
Turkish control chromosomes (Table 1; Table S5). This single base
substitution produced a missense mutation (GLU7ALA) at codon
7ofUCHL1 (NM_004181) (Fig. 1E), a position that is completely
conserved among vertebrate orthologs and is predicted to be
within the ubiquitin binding domain of the protein. The variant
was conﬁrmed to be homozygous in all three affected subjects,
and neither parent nor unaffected siblings were homozygous for
the variant by Sanger sequencing (Fig. 1F;Fig. S3). The cose-
gregation of this rare homozygous mutation with the neuro-
degeneration phenotype supported the hypothesis that it is the
cause of this Mendelian trait.
Is Predicted to Interfere with Substrate Binding. UCHL1
is a de-ubiquitinating enzyme, involved in the recycling of free
ubiquitin and therefore cytoplasmic protein degradation through its
C-terminal hydrolase enzymatic activity. To gain further insight into
the role of UCHL1 in neurodegeneration, we examined the struc-
tural implications and functional consequences of the GLU7ALA
mutation. The tertiary structure of UCHL1 resembles that of the
papain family (12) and is characterized by the presence of a cross-
over loop, termed L8, that spans and thereby restricts access to the
catalytic cleft that consists of the histidine-cysteine-aspartic acid triad
(12, 13) (Fig. 2A). In UCHL1, the L8 loop is short, thus requiring the
ubiquitin substrate to tunnel underneath it to achieve proteolysis;
residue E7 is located directly at the threshold of this tunnel (12, 13).
In the complex of UCHL1 and the ubiquitin substrate mimic,
ubiquitin vinyl methyl ester (UbVMe) (13), GLU7 is involved in
a hydrogen-bonding network that interacts both with the ubiquitin
substrate mimic and the L8 loop (Fig. 2A). The GLU7ALA variant is
therefore predicted to restrict the proper positioning of the substrate
for tunneling underneath the L8 loop, without conferring any other
signiﬁcant effects on the overall tertiary structure, e.g., exhibiting
a circular dichroism (CD) spectrum virtually identical to WT.
Results in Reduced Binding Afﬁnity for Ubiquitin and
Severely Reduced Hydrolytic Activity. We next analyzed the effects
of the GLU7ALA mutation on ubiquitin binding and C-terminal
Table 1. Variants detected within shared homozygous segments
Chromosome Start* End* Variants
variants that cosegregate
with the phenotype
4 38,187,084 49,061,848 66 3 1 1
6 44,575,446 53,593,569 49 1 0 0
8 39,869,633 43,791,691 9 0 0 0
11 114,223,577 117,570,851 17 1 1 0
Variants that were not previously identiﬁed in dbsnp (build 131) and 1,000 genomes project databases and among 2,400 exomes of
European subjects sequenced at Yale.
Bilguvar et al. PNAS Early Edition
hydrolase enzymatic activity. In Escherichia coli, we expressed and
puriﬁed WT UCHL1 (UCHL1
) and mutants UCHL1
, and UCHL1
(a cysteine to serine sub-
stitution in position 90), which carries a mutation in the active
cleft consisting of the abovementioned triad and was previously
shown to interfere with the catalytic activity (12–14). First, the
binding of ubiquitin to these puriﬁed proteins was measured using
isothermal titration calorimetry, from which the K
ubiquitin binding to these proteins were calculated (Fig. 2C;Table
S6). For the WT protein, the K
was 85 ±31 nM, and similar K
were determined for the CYS90SER and ILE93MET versions. By
contrast, GLU7ALA had a much higher K
sevenfold decreased ubiquitin binding (Table 2).
Next, the enzymatic activity of the puriﬁed enzymes was ex-
amined. In terms of its effects on catalytic activity, in an assay
with ubiquitin-7-amido-4-methycoumarin (ubiquitin-AMC) as sub-
strate where ubiquitin-AMC cleavage was continuously monitored
and measured on a ﬂuorescence spectrometer, UCHL1
exhibited <10% hydrolase activity compared with WT, whereas
exhibited the previously reported ∼50% ac-
tivity (7); as expected, UCHL1
exhibited no hydrolase
activity (Fig. 2B). Overall, the efﬁciency of UCHL1
as measured by k
, was 100-fold reduced relative to WT and
It has previously been suggested that UCHL1 may exhibit
novel dimerization-dependent ubiquitin ligase activity in vitro
(6). In an attempt to determine the effects of the GLU7ALA
variant on the suggested ubiquitin ligase activity of UCHL1, we
assayed and tested the enzymatic activity as previously described
(6). Neither UCHL1
ligase activity and did not ubiquitinate α-synuclein. Further
studies are needed to characterize the complete role of UCHL1
in proteasomal protein degradation.
Our molecular and biochemical data link the homozygous
mutation, which severely compromises ubiquitin
binding and hydrolase activity, with an early-onset progressive
Fig. 2. Structural and functional studies of UCHL1 carrying the GLU7ALA mutation. (A) Structural mapping of GLU7ALA (E7A) onto the crystal structure of
UCHL1 in complex with the ubiquitin substrate mimic, UbVMe (13) (PDB ID: 3IFW). UCHL1 is colored cyan, with the L8 loop colored orange. UbVMe is colored
yellow. Residue E7 is colored red. (Inset) Close-up of the hydrogen-bonding network at the threshold of the substrate tunnel. Panel made using the program
Pymol (www.pymol.org). (B) Comparison of the enzymatic activity of WT UCHL1 and the variants I93M, C90S, and E7A. Real-time release of ﬂuorescent AMC is
shown for WT-UCHL1 (black), I93M (blue), E7A (red), and C90S (green). (C) Binding isotherms of the titration of WT-UCHL1 and respective mutants with
ubiquitin. Binding of ubiquitin (500 μM) to corresponding proteins (50 μM) is shown. (Upper) Raw data. (Lower) Integrated heat data as enthalpy as a function
of molar ratio of ligand to protein. The solid line in the lower panel represents the best curve ﬁt to the data by using a one-site binding model.
www.pnas.org/cgi/doi/10.1073/pnas.1222732110 Bilguvar et al.
degenerative syndrome affecting multiple pathways within the
nervous system including the optic system, cerebral cortex, cere-
bellum, and spinal cord. In addition, myokymia was observed in
affected subjects, consistent with UCHL1’s role in the neuro-
muscular junction (15). The ataxia and muscular phenotypes in
individuals with the homozygous UCHL1
consistent with the phenotypes of the gracile axonal dystrophy
(gad) mouse, harboring a spontaneous in-frame deletion in Uchl1
that results in a truncated protein, as well as those of the Uchl1
KO mice that lack the entire Uchl1 protein (15–17). The gad
mouse has been reported to suffer from a progressive neurolog-
ical phenotype characterized by development of tremor and
sensory ataxia at around 3 mo of age postnatally, followed by
motor ataxia and ultimately leading to mortality. Neuropatho-
logical ﬁndings included nerve ﬁber loss with astrocytic pro-
liferation and considerable axonal swellings in the gracile fascicles
of the spinal cord along with axonal degeneration and formation
of spheroid bodies in the nerve terminals (16, 18, 19). Similarly,
recently generated Uchl1 KO mice have been shown to suffer
from markedly impaired synaptic transmission at the neuromus-
cular junction accompanied by structural defects such as loss of
synaptic vesicles and accumulation of tubulovesicular structures
at the presynaptic nerve terminals in addition to denervation of
the muscles (15). The phenotypes of both of these Uchl1 mouse
models closely resemble that of our patients, with the exception of
lack of optic nerve degeneration in the Uchl1 KO mice, examined
at about 4 mo of age. Further analyses of these models at later
stages will be needed.
In 1998, a heterozygous missense variant (ILE93MET) in
UCHL1 was described in a German family with Parkinson disease
and suggested as the cause of autosomal dominant Parkinson
disease type 5 (PARK5, OMIM 613643) (7).The UCHL1
variant results in a 50% reduction in hydrolytic activity; therefore,
the heterozygous affected subjects are expected to have 75% of
normal activity. Subsequent studies failed to replicate the ILE93-
MET variant in familial and sporadic forms of Parkinson disease
and identiﬁed only one other common UCHL1 variant, a serine to
tyrosine (SER18TYR) polymorphism (20, 21). Although a pro-
tective effect for the UCHL1
variant against Parkinson
disease was initially suggested, several subsequent association
studies yielded conﬂicting results (22–24). In the family presented
here, neither the patients homozygous for the UCHL1
variant nor their heterozygous parents or siblings exhibited Par-
kinsonian features on neurological examination. This ﬁnding
might be due to phenotypic heterogeneity associated with the
different UCHL1 variants (heterozygous ILE93MET mutation
vs. homozygous GLU7ALA mutation).
In addition to these genetic data, UCHL1’s expression has
been shown to be down-regulated in the brains of patients with
both Parkinson and Alzheimer’s diseases (25). UCHL1 has been
detected in neuroﬁbrillary tangles in idiopathic Alzheimer’s
disease patients, with the levels of soluble UCHL1 being in-
versely proportional to the number of tangles (25). Finally, it has
been shown that UCHL1 activity is required for normal synaptic
function and may improve the retention of memory in a mouse
model of Alzheimer’s disease (8). Collectively, these studies
suggest an association between UCHL1 dysfunction and neuro-
degeneration, which is strengthened by our discovery of a muta-
tion that almost completely abolishes its hydrolytic activity and
interferes with ubiquitin binding in a family with a neurodegen-
erative syndrome. Our ﬁndings further demonstrate the funda-
mental importance of protein degradation and the ubiquitin
pathway in proper functioning of the nervous system. This ob-
servation is in agreement with the recent identiﬁcation of Ubiq-
uilin 2 (UBQLN2) mutations in another neurodegenerative syn-
drome, familial amyotrophic lateral sclerosis (ALS) (26). Loss of
UBQLN2 function leads to impaired protein degradation and
accumulation of UBQLN2-containing protein aggregates in spinal
motor neurons of ALS patients. Interestingly, UBQLN2 accu-
mulated in spinal cord inclusion bodies irrespective of the patients
carrying UBQLN2 mutations. A similar pathology is observed in
the hippocampi of patients with ALS and dementia, again with or
without UBQLN2 mutations (26), implying that impairment of
ubiquitin-dependent proteolysis has pleiotropic effects leading to
broad degeneration of the nervous system.
Our results strengthen the link between ubiquitin-dependent
proteolysis and nervous system maintenance and demonstrate
that loss of UCHL1 activity results in a childhood-onset neuro-
degenerative syndrome. The observed phenotype is progressive,
leading to diffuse cerebral and cerebellar atrophy with aging.
Identifying the normal targets of UCHL1 and their biochemical
functions will provide insight into the maintenance of nervous
Materials and Methods
Human Subjects. The study protocol was approved by the Yale Human In-
vestigation Committee (protocol no. 0908005592). Institutional review board
approval for genetic studies, along with written consent from all study
subjects, was obtained at the participating institution.
EMG and SSEP Studies. Intramuscular needle EMG studies were performed in
all three patients testing deltoid and gastrocnemius muscles using conven-
tional techniques. Conduction velocities and response amplitudes were
measured for both upper and lower peripheric motor (median, ulnar, pe-
roneal and tibial) and sensory (median, ulnar and sural) nerves. For SSEP
studies, scalp evoked potential responses were recorded at 100 ms following
right tibial and median nerve stimulation in all three patients.
MRI. All MRI examinations were performed in a 3T scanner (Trio, Siemens)
using 8-channels head coil. The DTI datasets were obtained and analyzed as
described before (9). Brieﬂy, high resolution cranial MRIs were performed
using the following sequences: sagittal TSE T2, axial SE T1, TSE T2, FLAIR
and diffusion, coronal TSE T2, high resolution 3D TurboFlair T2 and recon-
structions, sagittal 3D TurboFlash T1 and reconstructions, sagittal 3D SPACE
T2 and reconstructions, coronal optic nerve focused TSE - STIR, SE T1, DTI- FT,
axial GRE T2.
IlluminaGenotyping.Whole-genome genotypingof the samples was performed
on the Illumina Platform with Illumina Human 610K Quad Beadchips using
the manufacturer’s protocol (Illumina) and as previously described (2, 3, 11).
Whole-Exome Capture and Sequencing. Genomic DNA sample was captured on
a NimbleGen 2.1M human exome array version 1.0 (Roche Nimblegen, Inc.)
Table 2. Binding and thermodynamic parameters for UCHL1
) 0.188 ±0.006 0.017 ±0.005 ND 0.084 ±0.003
(nM)* 85 ±31 646 ±288 32 ±629±5
ND 2.8 ×10
Values shown are the average of three different experiments ±SD. k
, catalytic constant; K
constant; ND, not detectable.
values for all proteins were determined by isothermal titration calorimetry.
Bilguvar et al. PNAS Early Edition
with modiﬁcations to the manufacturer’s protocol (2–4, 11). Brieﬂy, after
quality assessment, the genomic DNA was sheared by sonication, the ends of
fragments were repaired, adaptors were ligated, and appropriately sized
fragments were selected using agarose gel electrophoresis. Precapture li-
gation mediated PCR was performed, puriﬁed DNA was hybridized to the
array, and after multiple washes, the DNA was eluted and ampliﬁed by
postcapture mediated PCR. The pre- and post- capture libraries were com-
pared by quantitative PCR for the determination of the relative fold en-
richment of the targeted sequences. Single-read cluster generation was
performed on the Cluster Station (Illumina) and the captured, puriﬁed, and
clonally ampliﬁed library was then sequenced on Genome Analyzer IIx. One
lane of single-read sequencing at a read length of 74 bases was performed
on Genome Analyzer IIx following the manufacturer’s protocol.
Analysis of the Whole-Exome Sequencing Data. The sequence reads obtained
were aligned to the human genome (hg18) using Maq (27) and BWA (28)
software as previously described (4). Perl scripts were used to calculate the
percentage alignment of the reads to the reference genome and the tar-
geted exome. Similarly, perl scripts were used for the detection of mismatch
frequencies and error positions. SAMtools (29) was used for the detection of
single nucleotide variations on the reads aligned with Maq. The indels were
detected on the reads aligned with BWA for its ability to allow for gaps
during the alignment. Shared homozygous segments of the affected indi-
viduals were detected using Plink software version 1.07 (30), and the var-
iants were ﬁltered for shared homozygosity. The variants were annotated
for novelty with comparison with dbSNP (build 131), 1000 Genomes data-
base (August 4, 2010 release) and previous exome sequencing experiments
performed by our human genomics groups.
Sanger Sequencing. The exons and exon-intron boundaries of UCHL1 were
determined using the UCSC Genome Browser (http://genome.ucsc.edu),
unique primers were designed using Sequencher 4.8 (Gene Codes) and were
synthesized by Invitrogen. The fragments were ampliﬁed using standard
PCR techniques and sequenced on ABI’s 9800 Fast Thermocyclers (Applied
CD Spectroscopy. To determine CD spectra, stock solutions of UCHL1
mutants were diluted to
aﬁnal concentration of 10 μM with 50 mM Tris-HCl, pH 7.4 buffer. CD
spectra were recorded by using an Applied Photophysics spectropolarimeter
in the wavelength scan mode. Typically, data were obtained as an average
of three scans in the wavelength region of 280–195 nm by using a quartz
cuvette of 0.1-cm path.
Isothermal Titration Calorimetry. Isothermal titration calorimetry (ITC) was
carried out by using a VP-ITC Microcal calorimeter (Microcal) at 24 °C. All
protein samples were extensively dialyzed against 50 mM Tris-HCl, pH 7.6.
Titrations consisted of 5-μL injections of ubiquitin (Boston Biochem) into the
sample cell containing the respective UCHL1, at time intervals of 4 min to
ensure each peak returned to baseline. Each UCHL1 sample was followed by
a background titration of an equal volume of ubiquitin being titrated into
a sample cell containing buffer only, to account for the heat of ubiquitin
dilution, which was subtracted from the ubiquitin-UCHL1 data. All data were
analyzed by using the program Origin, version 7.0, included with the system.
The data were ﬁtted with a one-site binding model. Binding constants and
thermodynamic parameters of three experiments (average ±SD) are given.
Enzymatic Activity Assay. Wild type UCHL1 and the GLU7ALA, ILE93MET, and
CYS90SER mutants were expressed in E.coli and puriﬁed as previously de-
scribed (14). Puriﬁed proteins were diluted into reaction buffer (50 mM Tris-
HCl, pH 7.4, 1 mM DTT, and 1 mM EDTA) in a 200 μlﬂuorescence cuvette to a
ﬁnal concentration of 3 nM. Ubiquitin-AMC was added to the reaction
mixture to yield a ﬁnal concentration of 2500 nM to initiate the enzymatic
reaction. AMC cleavage was monitored at 25 °C on a PTI QuantaMaster
ﬂuorescence spectrometer with excitation at 380 nm and emission at
ACKNOWLEDGMENTS. We thank thepatients and families who contributed to
this study. This work was supported by the Yale Program on Neurogenetics,
National Institutes of Health (NIH) Grants RC2 NS070477 (to M.G.) and UL1
RR024139NIH (to S.M.), Yale Center for Mendelian Disorders Grant
U54HG006504 (to R.P.L., M.G., and S.M.), and the Gregory M. Kiez and Mehmet
Kutman Foundation. R.P.L. and A.L.H. are Investigators of the Howard Hughes
Medical Institute. SNP genotyping was supported in part by NIH Neuroscience
Microarray Consortium Award U24 NS051869-02S1 (to S.M.).
1. Ballard C, et al. (2011) Alzheimer’s disease. Lancet 377(9770):1019–1031.
2. Barak T, et al. (2011) Recessive LAMC3 mutations cause malformations of occipital
cortical development. Nat Genet 43(6):590–594.
3. Bilgüvar K, et al. (2010) Whole-exome sequencing identiﬁes recessive WDR62 muta-
tions in severe brain malformations. Nature 467(7312):207–210.
4. Choi M, et al. (2009) Genetic diagnosis by whole exome capture and massively parallel
DNA sequencing. Proc Natl Acad Sci USA 106(45):19096–19101.
5. Wilkinson KD, et al. (1989) The neuron-speciﬁc protein PGP 9.5 is a ubiquitin carboxyl-
terminal hydrolase. Science 246(4930):670–673.
6. Liu Y, Fallon L, Lashuel HA, Liu Z, Lansbury PT, Jr. (2002) The UCH-L1 gene encodes
two opposing enzymatic activities that affect alpha-synuclein degradation and Par-
kinson’s disease susceptibility. Cell 111(2):209–218.
7. Leroy E, et al. (1998) The ubiquitin pathway in Parkinson’s disease. Nature 395(6701):
8. Gong B, et al. (2006) Ubiquitin hydrolase Uch-L1 rescues beta-amyloid-induced de-
creases in synaptic function and contextual memory. Cell 126(4):775–788.
9. Dinçer A, et al. (2011) Diffusion tensor imaging of Guillain-Mollaret triangle in pa-
tients with hypertrophic olivary degeneration. J Neuroimaging 21(2):145–151.
10. Salmela MB, Cauley KA, Nickerson JP, Koski CJ, Filippi CG (2010) Magnetic resonance
diffusion tensor imaging (MRDTI) and tractography in children with septo-optic
dysplasia. Pediatr Radiol 40(5):708–713.
11. Bakircioglu M, et al. (2011) The essential role of centrosomal NDE1 in human cerebral
cortex neurogenesis. Am J Hum Genet 88(5):523–535.
12. Das C, et al. (2006) Structural basis for conformational plasticity of the Parkinson’s
disease-associated ubiquitin hydrolase UCH-L1. Proc Natl Acad Sci USA 103(12):
13. Boudreaux DA, Maiti TK, Davies CW, Das C (2010) Ubiquitin vinyl methyl ester binding
orients the misaligned active site of the ubiquitin hydrolase UCHL1 into productive
conformation. Proc Natl Acad Sci USA 107(20):9117–9122.
14. Larsen CN, Price JS, Wilkinson KD (1996) Substrate binding and catalysis by ubiquitin
C-terminal hydrolases: Identiﬁcation of two active site residues. Biochemistry 35(21):
15. Chen F, Sugiura Y, Myers KG, Liu Y, Lin W (2010) Ubiquitin carboxyl-terminal hy-
drolase L1 is required for maintaining the structure and function of the neuromus-
cular junction. Proc Natl Acad Sci USA 107(4):1636–1641.
16. Saigoh K, et al. (1999) Intragenic deletion in the gene encoding ubiquitin carboxy-
terminal hydrolase in gad mice. Nat Genet 23(1):47–51.
17. Yamazaki K, et al. (1988) Gracile axonal dystrophy (GAD), a new neurological mutant
in the mouse. Proc Soc Exp Biol Med 187(2):209–215.
18. Kikuchi T, Mukoyama M, Yamazaki K, Moriya H (1990) Axonal degeneration of as-
cending sensory neurons in gracile axonal dystrophy mutant mouse. Acta Neuro-
19. Mukoyama M, Yamazaki K, Kikuchi T, Tomita T (1989) Neuropathology of gracile
axonal dystrophy (GAD) mouse. An animal model of central distal axonopathy in
primary sensory neurons. Acta Neuropathol 79(3):294–299.
20. Lincoln S, et al. (1999) Low frequency of pathogenic mutations in the ubiquitin car-
boxy-terminal hydrolase gene in familial Parkinson’s disease. Neuroreport 10(2):
21. Wintermeyer P, et al. (2000) Mutation analysis and association studies of the UCHL1
gene in German Parkinson’s disease patients. Neuroreport 11(10):2079–2082.
22. Healy DG, et al. (2006) UCHL-1 is not a Parkinson’s disease susceptibility gene. Ann
23. Maraganore DM, et al.; UCHL1 Global Genetics Consortium (2004) UCHL1 is a Par-
kinson’s disease susceptibility gene. Ann Neurol 55(4):512–521.
24. Ragland M, Hutter C, Zabetian C, Edwards K (2009) Association between the ubiquitin
carboxyl-terminal esterase L1 gene (UCHL1) S18Y variant and Parkinson’s Disease: A
HuGE review and meta-analysis. Am J Epidemiol 170(11):1344–1357.
25. Choi J, et al. (2004) Oxidative modiﬁcations and down-regulation of ubiquitin car-
boxyl-terminal hydrolase L1 associated with idiopathic Parkinson’s and Alzheimer’s
diseases. J Biol Chem 279(13):13256–13264.
26. Deng HX, et al. (2011) Mutations in UBQLN2 cause dominant X-linked juvenile and
adult-onset ALS and ALS/dementia. Nature 477(7363):211–215.
27. Li H, Ruan J, Durbin R (2008) Mapping short DNA sequencing reads and calling var-
iants using mapping quality scores. Genome Res 18(11):1851–1858.
28. Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows-Wheeler
transform. Bioinformatics 25(14):1754–1760.
29. Li H, et al.; 1000 Genome Project Data Processing Subgroup (2009) The Sequence
Alignment/Map format and SAMtools. Bioinformatics 25(16):2078–2079.
30. Purcell S, et al. (2007) PLINK: A tool set for whole-genome association and pop-
ulation-based linkage analyses. Am J Hum Genet 81(3):559–575.
www.pnas.org/cgi/doi/10.1073/pnas.1222732110 Bilguvar et al.