Chorein detection for the diagnosis of chorea‐acanthocytosis

Abstract

Chorea-acanthocytosis (ChAc) is a severe, neurodegenerative disorder that shares clinical features with Huntington's disease and McLeod syndrome. It is caused by mutations in VPS13A, which encodes a large protein called chorein. Using antichorein antisera, we found expression of chorein in all human cells analyzed. However, chorein expression was absent or noticeably reduced in ChAc patient cells, but not McLeod syndrome and Huntington's disease cells. This suggests that loss of chorein expression is a diagnostic feature of ChAc. Ann Neurol 2004;56:299–302

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Chorein Detection for
the Diagnosis of
Chorea-Acanthocytosis
Carol Dobson-Stone, PhD,1Antonio Velayos-Baeza, PhD,1
Lea A. Filippone,2Sarah Westbury,3
Alexander Storch, MD,4Torsten Erdmann, MD,5
Stephen J. Wroe, MD, FRCP,6Klaus L. Leenders, MD,7
Anthony E. Lang, MD, FRCP,8Maria Teresa Dotti, MD,9
Antonio Federico, MD,9
Saidi A. Mohiddin, MD, MRCP,10
Lameh Fananapazir, MD, FRCP,10Geoff Daniels, PhD,11
Adrian Danek, MD,12and Anthony P. Monaco, MD1
Chorea-acanthocytosis (ChAc) is a severe, neurodegenera-
tive disorder that shares clinical features with Hunting-
ton’s disease and McLeod syndrome. It is caused by mu-
tations in VPS13A, which encodes a large protein called
chorein. Using antichorein antisera, we found expression
of chorein in all human cells analyzed. However, chorein
expression was absent or noticeably reduced in ChAc pa-
tient cells, but not McLeod syndrome and Huntington’s
disease cells. This suggests that loss of chorein expression
is a diagnostic feature of ChAc.
Ann Neurol 2004;56:299–302
Chorea-acanthocytosis (ChAc, OMIM 200150) is an
autosomal recessive neurodegenerative disorder charac-
terized by progressive onset of hyperkinetic movements
and unusual spiny erythrocyte morphology (acanthocy-
tosis).1ChAc diagnosis can be challenging, due to in-
terlaboratory variability in acanthocyte detection effi-
ciency,2
and the clinical similarity of ChAc to
Huntington’s disease (HD) and McLeod syndrome
(MLS, OMIM *314850), an X-linked neuroacanthocy-
tosis.3
CHAC, encoding the large (?3,000 amino acids)
protein chorein, is the gene mutated in chorea-
acanthocytosis.4,5CHAC, now renamed VPS13A,6is
organized into 73 exons and has two main splice
forms: isoform 1A (exons 1–68, 70–73) and isoform
1B (exons 1–69).4Because of the size of the gene and
the allelic heterogeneity of ChAc,7mutation screening
of VPS13A is a cumbersome process, impeding diag-
nostic progress. Herein, we report the generation of an-
tichorein polyclonal antisera. We note the apparently
ubiquitous distribution of chorein and demonstrate
that loss of chorein expression in erythrocyte mem-
branes and other cells is a diagnostic feature of ChAc.
Subjects and Methods
Subjects
Informed patient consent for molecular analysis of blood
samples was obtained according to local guidelines. Samples
from 14 patients with a clinical diagnosis of chorea-
acanthocytosis (10 men, 4 women) were studied. Patients 1
to 4 correspond to CHAC2IV5, CHAC6II1 and a newly
diagnosed brother, and CHAC11II2 in previous studies4,8;
Patients 5, 6, 7, and 14 correspond to probands 24, 11, 2,
and 23, respectively, in a previous study.7All displayed char-
acteristic clinical features of chorea-acanthocytosis1that in-
cluded elevation of serum creatine kinase (14 of 14 patients),
ankle areflexia (10/14), cognitive or neuropsychiatric changes
(12/14), limb chorea (13/14), orofacial dyskinesia (including
tongue dystonia and lip biting, 12/14), dysarthria (11/14),
involuntary vocalizations (7/14), and parkinsonian features
(2/14). Nine of the 14 patients had experienced seizures.
Blood samples from two men with MLS were also studied
(Patients 3 and 13 from Danek and colleagues3).
Mutation Detection
ChAc patient DNA was screened for VPS13A mutations as
described previously.7At least one heterozygous VPS13A
mutation likely to cause disease was found in each ChAc
patient, as shown in the Table.
Production of Polyclonal Antiserum Anti-chor1
A fragment of chorein comprising amino acids 27 to 326
wasexpressedasafusion
S-transferase (GST-chor1). Bacterial expression and purifica-
tion of GST-chor1 was performed as described by Frangioni
and Neel,9using glutathione Sepharose 4B beads (Amersham
Biosciences UK Ltd, Chalfont St. Giles, UK). Immunization
of rabbits with the chor1 moiety was performed by Eurogen-
tec Bel SA (Herstal, Belgium); the serum collected 87 days
after immunization was designated anti-chor1.
proteinwithglutathione
Western Blotting of Cell Lysates
Cells were harvested by trypsinization and centrifugation at
3,400g for 5 minutes. Cell pellets were washed with ice-cold
phosphate-buffered saline then lysed in 10 ? volume of ice-
From the1Wellcome Trust Centre for Human Genetics, University
of Oxford, Oxford, UK;2Molecular Biology and Genetics, Cornell
University, Ithaca, NY;3St. John’s College, University of Oxford,
Oxford, UK;4Department of Neurology, Technical University of
Dresden, Dresden, Germany;5Department of Neurology, Vinzenz
von Paul Hospital–Rottenmu ¨nster, Rottweil, Germany;
ment of Clinical Neurology, Ipswich Hospital, Ipswich, UK;7Gro-
ningen University Hospital, Groningen, The Netherlands;8Division
of Neurology, University of Toronto, Toronto, Canada;9Depart-
ment of Neurological and Behavioural Sciences, Section of Neurol-
ogy and Neurometabolic Diseases, Medical School, University of
Siena, Siena, Italy;10Cardiovascular Branch, National Heart Lung
and Blood Institute, Bethesda, MD;11Bristol Institute for Transfu-
sion Sciences, Bristol, UK; and
Maximilians-Universita ¨t, Munich, Germany.
6Depart-
12Neurologische Klinik, Ludwig-
Received Mar 17, 2004, and in revised form May 24. Accepted for
publication May 25, 2004.
Published
(www.interscience.wiley.com). DOI: 10.1002/ana.20200
onlineJul27,2004,inWileyInterScience
Address correspondence to Prof Monaco, The Wellcome Trust Cen-
tre for Human Genetics, University of Oxford, Roosevelt Drive,
Headington, Oxford, OX3 7BN, UK.
E-mail: anthony.monaco@well.ox.ac.uk
© 2004 American Neurological Association
299
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cold lysis buffer (50mM Tris-HCL, pH 8.0, 150mM NaCl,
1% Triton X-100, and 1 ? protease inhibitor cocktail;
Sigma, Poole, UK). After 20-minute incubation on ice, ly-
sates were clarified by centrifugation at 16,000g at 4°C for
20 minutes. Samples containing equal quantities of protein
(20?g) were denatured and loaded on a NuPAGE 3 to 8%
Tris-acetate gel (Invitrogen, Paisley, UK), followed by elec-
trophoretic transfer on to polyvinylidene difluoride mem-
branes. Immunodetection was performed according to stan-
dard protocols,10
using anti-chor1 antiserum (1:5,000
dilution), preimmune serum (1:5,000) or mouse anti–hun-
tingtin antibody (1:10,000; Serotec Ltd, Oxford, UK) fol-
lowed by peroxidase-conjugated goat anti–rabbit or anti–
mouse antibody (1:5,000; Bio-Rad Laboratories Ltd, Hemel
Hempstead, UK). Proteins were visualized using the ECL
plus Western Blotting Detection System (Amersham). For
antibody depletion experiments, anti-chor1 antiserum was
diluted 1:2,500 in 10mM Tris-HCL, pH 7.4, 0.8% NaCl.
Half was passed three times through an affinity column con-
taining chor1-conjugated agarose, and half was left un-
treated. Both serum preparations were subsequently adjusted
to the appropriate dilution for use in Western blotting.
Western Blotting of Erythrocyte Membranes
Erythrocyte membranes were prepared according to Dodge
and colleagues,11with minor modifications. In brief, 10ml of
ice-cold erythrocyte wash solution (5mM Na2HPO4, pH
8.0, 0.9% NaCl, 1 ? protease inhibitor cocktail) were added
to 2.5ml thawed whole blood and subjected to centrifugation
at 3,400g, 4°C for 10 minutes. Any intact erythrocytes were
lysed by resuspension of the pellet in 2ml erythrocyte lysis
solution (5mM Na2HPO4, pH 8.0, 1 ? protease inhibitor
cocktail) followed by centrifugation at 16,000g for 5 min-
utes. The loose pellet of membranes was washed multiple
times in erythrocyte lysis solution until the supernatant re-
mained colorless. Membranes (2.5?l thereof) of were sub-
jected to electrophoresis and blotted as above.
Results
Detection of Endogenous Chorein
Western blot analysis of ChAc Patient 1 and control
lymphoblastoid cell lysates shows that anti-chor1 serum
recognizes chorein (see Fig 1A). A band similar in size
to huntingtin (350kDa, lanes 7 and 8) was detected in
control cells (lane 5); this is consistent with the pre-
dicted molecular weight of chorein (360kDa). This
band was absent in cells from Patient 1 (lane 6). The
signal was not detected using preimmune serum (lanes
1 and 2) or when using serum depleted in chor1-
binding antibodies (lanes 3 and 4), thereby confirming
the detection specificity. To investigate chorein distri-
bution, we analyzed lysates from a variety of cell lines
routinely used in tissue culture techniques by Western
blot (see Fig 1B). Anti-chor1 antiserum detected a
high-molecular-weight signal in all cell lines analyzed.
Expression of Mutant Chorein in
Chorea-Acanthocytosis Cell Lines
Western blot analysis of lymphoblastoid cell lysates
shows that ChAc Patients 2 to 4 have markedly re-
duced chorein levels compared with the control (Fig
2A, see Table), in contrast with unaffected family
members (lanes 3, 5, and 6) and MLS Patient 1 (lane
Table. ChAc and McLeod Syndrome Patients Analyzed in This Study
Patienta
DNA Changeb
Protein Changeb
Type of Mutation
Tissue
analyzed
Chorein
Expressionc
VPSI3A mutations
1 (4)
2, 3 (4)
4 (4)
5 (7)
6 (7)
7 (7)
8
9
10
11
12
13
14 (7)
XK mutations
M1 (13)
M2 (3)
[1592del]
[4354T?C]
[237del]
[6419C?G]
[1125_1128del]
[2288 ? 2T?C]
[1596 ? 2A?C]d
[1596 ? 1G?C]d
[EX46_EX50del]
[5909_5910del]
[188-5T?G]
[495 ? 1G?A]
[1208_1211del]
[1592del]
[4354T?C]
[9429_9432del]
[9190del]
[?]
[8472-1G?C]
[8907 ? 2T?A]d
[4355C?G]d
[EX46_EX50del]
[?]
[?]
[?]
[7867C?T]
[1531fsX6]
[S1452P]
[E80fsX10]
[S2140X]
[S375fsX22] [?]
[SD]
[SA]
[SA]
[unknown]
[E1970fsX3] [?]
[SA]
[SD]
[Q403fsX5] [R2623X]
[1531fsX6]
[S1452P]
[R3143fsX4]
[V3064fsX16]
Frameshift
Missense
Frameshift
Nonsense
Frameshift
Splicing
Splicing
Splicing
Deletion
Frameshift
Splicing
Splicing
Frameshift
Frameshift
Missense
Frameshift
Frameshift
?
Splicing
Splicing
Nonsense
Deletion
?
?
?
?
Lb
Lb
Lb
Fb
Fb
Fb
Ec
Ec
Ec
Ec
Ec
Ec
Ec
?
?
?
?
?
?
?
?
?
?
?
?
?
[SA]
[SD]
[S1452X]
[unknown]
[?]
[?]
508 ? 1G?A
EX1del
SD
unknown
Splicing
Deletion
Lb
Ec
???
???
aReferences for patients who were screened for mutations in a previous study are given in parentheses.
bNucleotides and amino acids are numbered according to the cDNA sequence of VPS13A/CHAC isoform A reported by Rampoldi and
colleagues4(GenBank accession no. NM_033305) or that of XK13(GenBank accession no. Z32684). Mutation nomenclature is as recom-
mended by the Human Genome Variation Society (http://www.genomic.unimelb.edu.au/mdi/mutnomen/index.html).
cChorein expression levels are denoted as follows: - ? undetectable; ? ? markedly reduced compared with control; ??? ? similar to control
levels.
dMutations detected in this study that were identified in another family previously7
MI ? McLeod syndrome patient 1; SD ? splice donor; SA ? splice acceptor; ? ? second heterozygous mutation not found; Lb ? lympho-
blastoid cell line; Fb ? primary skin fibroblast; Ec ? erythrocyte membrane.
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8). The affected son in Family CHAC11 (Patient 4)
has inherited a maternal 237del mutation in exon 4
and a paternal 9429_9432del mutation in exon 72.
Any protein that is produced from the 237del allele
will be severely truncated (predicted size 10kDa). The
weak signal seen for Patient 4 therefore must derive
from truncated protein generated by the 9429_9432del
allele (357kDa), and/or other chorein isoforms, such as
isoform 1B (345kDa), which is theoretically unaffected
by the exon 72 mutation. Chorein expression in pri-
mary skin fibroblasts from ChAc Patients 5 to 7 was
undetectable or markedly reduced (see Table).
Expression of Chorein in Erythrocyte Membranes
Western blot analysis of erythrocyte membrane frac-
tions from ChAc Patients 8 to 14, MLS Patient 2, and
two healthy individuals is shown in Figure 2B.
Chorein expression was undetectable or markedly re-
duced in all ChAc patients analyzed. In contrast, a sig-
Fig 2. Analysis of chorein expression in ChAc and MLS pa-
tient cells. (A) Analysis of lymphoblastoid cell lines. Twenty-
microgram protein samples were separated by sodium dodecyl
sulfate polyacrylamide gel electrophoresis and analyzed by West-
ern blot. Cells were derived from ChAc patients (P1–7, lanes
1, 2, 4, 7), unaffected members of ChAc families (lanes 3, 5,
6), a McLeod syndrome patient (M1, lane 8), and a healthy
control (C, lane 9). The VPS13A genotype is indicated above
CHAC6 and CHAC11 family members (?, wild type; ?,
mutant allele). Blot stripping and immunodetection of EEA1
demonstrated equal loading of samples (data not shown). (B)
Analysis of erythrocyte membranes. Membranes were prepared
from ChAc Patients 8 to 14 (P8–14, lanes 1–7); MLS Pa-
tient 2 (M2, lane 8); and healthy controls (C1, C2, lanes 9,
10). Blood had been stored at ?20°C for different lengths of
time, ranging from 8 weeks 4 days (C2) to 159 weeks 5 days
(P14). M2 blood had been stored at ?192°C for 654 weeks
3 days. An arrow points to the band corresponding to chorein;
a lower, cross-reacting band (asterisk) possibly corresponds to
spectrin, the most abundant protein associated with the eryth-
rocyte membrane.14Immunodetection with anti–spectrin anti-
body supports this hypothesis (data not shown). Total protein
staining of the blot with 0.1% Ponceau S solution before im-
munodetection demonstrated equal loading of samples (data
not shown).
Fig 1. Detection of chorein expression. (A) Anti-chor1 anti-
serum detects endogenous chorein. Twenty-microgram protein
samples from lymphoblastoid cell lines (C ? healthy control,
P1 ? ChAc Patient 1) were separated by sodium dodecyl sul-
fate polyacrylamide gel electrophoresis and analyzed by Western
blot. Blots were analyzed with preimmune serum (lanes 1 and
2) or anti-chor1 antiserum (lanes 3–6). Anti-chor1 antiserum
was either run through a column bound with chor1 antigen
to remove chor1-binding antibodies (column-treated ?, lanes
3, 4) or left untreated (column-treated ?, lanes 5, 6). An
anti-huntingtin blot (anti-Htt, lanes 7, 8) is provided for size
comparison (Htt ? 350kDa). Because of their similar size, it
is not possible to distinguish between chorein isoforms 1A
(360kDa) and 1B (345kDa). (B) Expression of chorein in
different cell types. Human cell lines were derived from cervi-
cal carcinoma (HeLa), fetal lung (MRC5), embryonic kidney
(293T), lymphoblasts (lymph), neuroglioma (H4), hepatocarci-
noma (Hep3B), myelogenous leukemia (K562), and rhabdo-
myosarcoma (RD). Nonhuman cell lines were derived from
African green monkey kidney (COS-7) and Chinese hamster
ovary (CHO-KI). The much weaker signal in nonhuman cell
lines is presumably caused either by a reduction in chorein
ortholog expression or by limited cross-reaction with the anti-
chor1 antiserum. Blot stripping and immunodetection of early
endosome antigen 1 (EEA1) demonstrated equal loading of
samples (data not shown).
Dobson-Stone et al: Chorein Detection in ChAc
301

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nal comparable in strength with the controls was ob-
served in MLS Patient 2 (lane 8), even though blood
from this patient had been stored for over 12 years.
Membrane fractions from two HD patients also
showed normal levels of chorein expression (data not
shown).
Discussion
Mutations in the VPS13A gene are associated with the
severe neurodegenerative disorder chorea-acanthocytosis.
In this study, we report the first cellular detection of
chorein, the VPS13A gene product. We found that
chorein is expressed in cell lines derived from a wide
variety of human tissues, as well as primary skin fibro-
blasts and erythrocytes. This is supported by previous
analyses that showed ubiquitous expression of VPS13A
mRNA.4
We have demonstrated that 19 different VPS13A
mutations, including one missense mutation, lead to
absence or marked reduction of chorein expression in
ChAc patients. Even in a patient with an isoform 1A–
specific mutation (9429_9432del), very little chorein
expression was observed. We observed previously that
several patients with ChAc harbored isoform 1A–spe-
cific mutations and speculated that exons 70 to 73
were essential for some functions of chorein.7The rel-
atively poor expression of isoforms lacking these exons
also may explain their inability to compensate for full-
length chorein in ChAc.
We have shown that chorein can be detected in as-
sociation with the erythrocyte membrane (see Fig 2B).
MLS and HD patient samples show normal chorein
expression levels, in contrast with ChAc patients. This
has obvious implications for ChAc diagnosis. Cur-
rently, if the VPS13A gene has not been screened,
ChAc can be diagnosed only by excluding other clini-
cally similar disorders. As VPS13A is a large gene with
many exons, screening is costly and time consuming.
Western blotting of patient erythrocyte membranes
perhaps could give an early indication of the disorder
before a precise diagnosis is given by a VPS13A gene
screen.
Although chorein was absent or markedly reduced in
all patient erythrocytes analyzed so far, one cannot ex-
clude ChAc as a diagnosis if chorein is present in a
sample. The missense mutation S1452P appears simply
to affect chorein dosage (see Fig 2A); however, some
substitutions may lead to apparently normal levels of
chorein that is nevertheless functionally defective. Also,
some mutations may allow almost normal expression
levels of mutant chorein lacking only a few exons,
which might not be resolved from wild-type chorein
because of its large size. However, we anticipate that
this would not be a significant problem, because mis-
sense changes compose less than 7% (5/75) of VPS13A
mutations described so far (previous studies4,5,7and
this study), and most transcripts containing premature
stop codons are believed to be rapidly degraded.12In
any case, if chorein is absent in a patient sample, the
most likely diagnosis will be chorea-acanthocytosis.
This work was supported by the Wellcome Trust (060886/Z/00/
Z,C.D.-S., 045093/Z195, A.P.M.) and the Marie Curie postdoc-
toral fellowship (QLGA-CT-2001-51850, A.V.-B.).
We thank the patients and their families for their participation, L.
Lonie for denaturing high-performance liquid chromatography anal-
ysis, and all colleagues who contributed patient samples, notably S.
Carre `, X. Ferrer, A. Lees, A. Ne ´meth, J. Palace, G. Rudolf, and C.
Verellen.
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