вход по аккаунту


Characterization of PLA2G6 as a locus for dystonia-parkinsonism.

код для вставкиСкачать
Characterization of PLA2G6 as a Locus for
Coro Paisan-Ruiz, PhD,1,2 Kailash P. Bhatia, FRCP,3 Abi Li,2 Dena Hernandez, MS,1 Mary Davis, PhD,2
Nick W. Wood, PhD, FMedSci,2 John Hardy, PhD, FMedSci,1,2 Henry Houlden, PhD,2
Andrew Singleton, PhD,1 and Susanne A. Schneider, MD3
Background: Although many recessive loci causing parkinsonism dystonia have been identified, these do not explain all cases of
the disorder.
Methods: We used homozygosity mapping and mutational analysis in three individuals from two unrelated families who presented with adult-onset levodopa-responsive dystonia-parkinsonism, pyramidal signs and cognitive/psychiatric features, and cerebral and cerebellar atrophy on magnetic resonance imaging but absent iron in the basal ganglia.
Results: We identified areas of homozygosity on chromosome 22 and, subsequently, PLA2G6 mutations.
Interpretation: PLA2G6 mutations are associated with infantile neuroaxonal dystrophy and have been reported previously to
cause early cerebellar signs, and the syndrome was classified as neurodegeneration with brain iron accumulation (type 2). Our
cases have neither of these previously pathognomic features. Thus, mutations in PLA2G6 should additionally be considered in
patients with adult-onset dystonia-parkinsonism even with absent iron on brain imaging.
Ann Neurol 2009;65:19 –23
In patients with recessive dystonia-parkinsonism, the
differential diagnosis is complex and includes pantothenate kinase–associated neurodegeneration (PKAN, also
called Hallervorden–Spatz disease or neurodegeneration
with brain iron accumulation [NBIA] type 1) caused
by mutations in PANK2 on chromosome 20 (OMIM
234200),1 Kufor–Rakeb syndrome caused by mutations in ATP13A22 on chromosome 1p (OMIM
606693), and DYT16 linked to mutations in PRKRA
on chromosome 2.3 It may also include the parkinsonism loci PARK24 (chromosome 6q; OMIM 602544),
PARK65 (chromosome 1p; OMIM 608309), PARK76
(chromosome 1p; OMIM 602533) and the L-dopa–responsive dystonias. In addition, it is likely that there
will be other loci for this syndrome.
To find the causes of parkinsonism dystonia in consanguineous families, to better group them for clinical
characterization, and to identify any families who do
not have mutations in the known loci, we have performed systematic screening using whole-genome genotyping methods. Families showing homozygosity at the
different loci were then sequenced to find the homozygous gene mutations.
Here, we identified homozygosity on chromosome
22 and, subsequently, PLA2G6 mutations in two
families. These individuals did not fit the previously
described phenotype of this syndrome, infantile neuroaxonal dystrophy (INAD), which is clinically characterized by mental retardation, early cerebellar degeneration, pyramidal signs, and visual disturbances.7 Our
finding demonstrates that PLA2G6 mutations should
be considered in patients with adult-onset dystonia
parkinsonism, even in the absence of iron deposition
on magnetic resonance imaging (MRI).
From the 1Laboratory of Neurogenetics, National Institute on Aging, Intramural Research Program, National Institutes of Health,
Bethesda, MD; and 2Department of Molecular Neuroscience and
Sobell Department of Motor Neuroscience and Movement Disorders, Institute of Neurology, University College London, London,
United Kingdom.
Potential conflict of interest: Nothing to report.
Address correspondence to Dr Bhatia, Sobell Department of Motor
Neuroscience and Movement Disorders, Institute of Neurology, Box
13, UCL, Queen Square, London WC1N 3BG, United Kingdom.
Subjects and Methods
Ethical/institutional review board committee approval was
obtained at our institutions. We started with 10 individuals
(8 affected and 2 unaffected) from 5 unrelated consanguineous families affected by an atypical akinetic rigid syndrome
who were sampled. Ten milliliters of venous blood was taken
and DNA extracted by conventional methods.
Genotyping was carried out using either Infinium II HumanHap317 BeadChips or Infinium II HumanHap240
Additional Supporting Information may be found in the online version of this article.
Received Feb 12, 2008, and in revised form Mar 18. Accepted for
publication Apr 4, 2008.
Published online June 20, 2008, in Wiley InterScience
( DOI: 10.1002/ana.21415
© 2008 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
Fig 1. (A) Pedigree of Family 1 (p.R741Q mutation) showing
the segregation. (B) Pedigree of Family 2 (p.R747W mutation). Solid symbols represent affected individuals; open symbols represent unaffected family members; diamond symbols
indicate (unaffected) individuals of unspecified sex. #Further
siblings. The index cases are highlighted by an arrow. Double
lines indicate consanguineous marriages between first cousins.
⫹/⫹, mutant; ⫺/⫹, carrier; ⫺/⫺, wild type. Neither mutation variant was found in 226 control individuals of ethnic
Indian/Pakistani origin.
BeadChips (Illumina, San Diego, CA.), and homozygosity
mapping was performed as previously described.3
PLA2G6 Mutation Screening
In the three families who showed homozygosity, all 17 coding exons and at least 50bp of flanking intronic sequences
were analyzed by performing polymerase chain reaction analysis using 10pmol of both forward and reverse genomic
primers (sequence available on request) as described previously.3
Mutation Analysis
Five affected members from three of the five families
shared a homozygous region of chromosome 22q12.322q13.1, which contains the PLA2G6 gene. The restricted area compiling 320 consecutive single nucleotide polymorphisms is flanked by rs2076083 and
rs135737 single nucleotide polymorphisms and was delimited by one affected member from Family 1.
Sequencing the coding region of PLA2G6 demonstrated the presence of different novel mutations in affected members from Families 1 and 2. In Family 1,
two affected members and six unaffected members
were available for genetic testing. A homozygous mutation at position c.2222G⬎A resulting in p.R741Q
was detected in both patients. Analysis of the p.R741Q
mutation in the unaffected individual showed segregation with the disease (Fig 1). In Family 2, for which
one affected individual was available for genetic testing,
homozygous c.2239C⬎T (p.R747W) mutation was
found. No other family members were available for
Annals of Neurology
Vol 65
No 1
January 2009
analysis. Neither variant was found in 186 Pakistani
individuals of the Human Genome Diversity Project
DNA panel ( or 40 samples of ethnic Indian/Pakistani origin.
Both mutations are conserved among species.
In Family 3, we detected a region of homozygosity
over 28.56Mb (Table 1) at chromosome 22, as well as
homozygous tracts in other areas of the genome. Analysis of PLA2G6 in affected individuals in Family 3 and
in the remaining two families (Families 4 and 5), without homozygosity at chromosome 22q12.3-22q13.1,
did not show variation in PLA2G6. We conclude that
the disease in these other three families is caused by
mutations at unknown loci.
Clinical Features of PLA2G6 Homozygotes
FAMILY 1. A summary of the clinical characteristics is
given in Table 2. A 34-year-old Indian woman (Table
2) from a consanguineous background (parents were
first cousins) had normal milestones. At age 26, over 6
months she developed a rapid cognitive decline, slow
movements, clumsiness, imbalance, hand tremor, and
dysarthria. At 27, she could not walk without assistance. Neuropsychological assessment showed severe,
widespread cognitive dysfunction (verbal intelligence
quotient 66, severe impairment of verbal and visual
memory [scores ⬍ 5th percentile], word retrieval difficulties, frontal executive dysfunction), and depression.
She had facial hypomimia, eyelid opening apraxia,
square wave jerks in the primary position, supranuclear
vertical gaze palsy, and hypometric vertical saccades.
Kayser–Fleischer rings and pigmentary retinopathy
were absent. She was dysarthric with slow tongue
movements. There was marked generalized rigidity and
dystonia in all limbs, a left pill rolling rest tremor, mild
postural arm tremor, and bilateral bradykinesia. Power
was normal with brisk reflexes with ankle clonus but
flexor plantar responses. Assisted gait was narrow
based, stiff-legged, and with extremely poor postural
reflexes. She tended to walk on her toes.
Investigations showed increased creatine kinase
(215IU/L; reference range, 24 –173IU/L). Cerebrospinal fluid analysis showed oligoclonal IgG and decreased
homovanillic acid but was otherwise normal. Normal
results included electrolytes, coagulation screen, serum
copper, ceruloplasmin, ferritin, glucose, liver/renal
function tests, serum amino acids, urinary organic acids, very long fatty acids, white cell enzymes, acanthocytes screen, vitamin B12 levels, antinuclear antibodies,
Treponema pallidum haemagglutination test, Venereal
Disease Research Laboratory, neutrophil cytoplasmatic
antibodies, and thyroid function tests. Gene tests were
normal for IT15, DRPLA, PARK2, SCA-1, -2, -3, -6,
-7, and ATP13A2. She had a normal phenylalanine
loading test, electroencephalogram, electromyogram,
Table 1. Chromosome Position of the Single Nucleotide Polymorphisms Flanking the Homozygous Chromosome
22 Region Found in the 3 Recessive Dystonia Parkinsonism Families
SNPs (rs number)
Localization (bp)
Recessive Dystonia
Family 1 (2 patients)
Family 2 (1 patient)
Family 3 (2 patients)
SNP ⫽ single nucleotide polymorphism.
nerve conduction studies, visual-evoked potentials, skin
biopsy, and bone marrow aspiration.
Brain MRI (Fig 2) showed generalized cerebral atrophy and frontal white signal changes. Absence of iron
deposition particularly in the basal ganglia was confirmed by T2* sequence. Spinal MRI was normal. A
DaT single-photon emission computed tomography
scan showed markedly reduced uptake bilaterally in the
basal ganglia.
Trihexyphenidyl was not beneficial, but L-dopa commenced at age 28 had an unsustained but initially dramatic response resulting in almost independent walking. However, she developed prominent dyskinesias.
At 29, her condition had deteriorated, with generalized bradykinesia and severe rigidity causing immobility. She had a staring expression and facial muscle
twitches with a factitious smile. At age 32, a percutan
endoscopic gastrostomy had to be inserted.
A cousin was similarly affected with gradual leg onset with dragging and dystonia at age 10. At age 26,
she developed arm and leg tremor, bradykinesia, and
became immobile. Similarly to her cousin, dopaminergic treatment was beneficial but caused prominent dyskinesias. However, little is known about this individual.
This 21-year-old man of Pakistani descent
was a product of double consanguinity (see Fig 1).
There was no family history of neurological disease.
Birth, early milestones, and childhood were normal,
and he attended university. At 18, he developed a fairly
rapid onset of dragging of his foot, cognitive decline,
and personality changes with aggression. He developed
urinary frequency and nocturia. Swallowing was normal.
At age 19, neuropsychological assessment demonstrated impaired intellectual function (verbal intelligence quotient 88, performance intelligence quotient
74), visual memory, nominal functions, and frontal executive dysfunction. He had blepharoclonus. Saccadic
pursuit was jerky. There was no supranuclear gaze
palsy or nystagmus or Kayser–Fleischer rings. There
were asymmetric pyramidal features with spasticity, hyperreflexia, marked ankle clonus and extrapyramidal
signs with cogwheeling, bradykinesia, and foot dystonia
(see Supplementary video). The gait was hemiparetic
with right leg circumduction and extensor axial dystonia. Postural reflexes were markedly impaired.
Blood tests were all normal. Brain MRI was unremarkable apart from mild generalized volume loss. Iron
deposition was excluded by T2* imaging (see Fig 2).
Spinal MRI was normal. A dopamine transporter
single-photon emission computed tomography scan
showed markedly reduced uptake in both striata. Cerebrospinal fluid examination was normal without oli-
Fig 2. Bain magnetic resonance imaging (MRI) of Patients 1
(A) and 2 (B). (A) There is no evidence of iron accumulation
in the basal ganglia (T2*-weighted image; left). There are
white matter increased signal changes, mainly around the
frontal horn (T2 fluid-attenuated inversion recovery; top right,
long arrow). There is progressive generalized volume loss. Cerebellar volume is normal (T1-weighted image; bottom right,
short arrow). (B) There is no iron deposition in the basal
ganglia on T2*-weighted imaging (left). A normal MRI study,
apart from mild cerebral atrophy (T1-weighted slides; right).
Paisan-Ruiz et al: PLA2G6-Dystonia-Parkinsonism
Table 2. Summary of Demographic and Clinical Findings
Demographics and Clinical Findings
Family 1
(Patient 1)
Current age (yr)
Family 1
(Patient 2)
Family 2
26/cognitive deterioration
10/foot drag
18/foot drag
Cognitive decline
Psychiatric features
l-dopa response
Age of onset (yr)/first symptom
Extrapyramidal features
l-dopa–induced dyskinesias
Eye movement abnormalities
Imbalance/impaired postural reflexes
Cerebellar signs
Sensory abnormalities
Autonomic involvement
Pyramidal signs
Tremor with rest component (pill-rolling).
l-dopa–naive, treated with a dopamine agonist.
NK ⫽ not known; NA ⫽ not applicable; ⫹ ⫽ mild; ⫹⫹ ⫽ severe.
goclonal bands. An electroencephalogram was normal.
Ropinirole introduced at age 20 resulted in marked improvement of gait, bradykinesia, and dystonia.
We studied five families with an atypical akinetic rigid
syndrome and describe a yet unrecognized phenotype
of PLA2G6 mutations that is adult-onset L-dopa–responsive complicated parkinsonism without brain iron
accumulation on MRI in two of these families. The
remaining families showed more extensive homozygosity tracts (Family 3) or no homozygosity of the area
that contains the PLA2G6 gene (Families 4 and 5).
Mutations in PLA2G6 also cause NBIA, and iron
was present in all PLA2G6-related NBIA cases described recently.8 In addition to marked cerebellar atrophy and progressive white matter changes, iron accumulation of the pallida (affecting medial and lateral
portions) was also detected in six further PLA2G6related INAD patients.9 In one, iron was not (yet)
present on T2-weighted imaging (T2* scans not presented) 2 years after disease onset but prominent on
both T2- and T2*-weighted MRI on 6-year follow-up.
Our finding of absent iron in the basal ganglia as confirmed by T2*-weighted imaging, up to 12 years after
Annals of Neurology
Vol 65
No 1
January 2009
disease duration, illustrates that a diagnosis of
PLA2G6-related neurodegeneration should not only be
considered in patients with dystonia-parkinsonism with
brain iron accumulation but also those without. Visualevoked potentials and electromyograms, which are typically abnormal in classic young-onset INAD,7 were
normal in our patient.
Pathologically, INAD is characterized by axonal degeneration with distended axons (spheroid bodies),
which stained ubiquitin-positive in PLA2G6-mouse
models.10 However, there is evidence for pathological
heterogeneity of INAD as cases with clinical and
pathological features of INAD negative for PLA2G6
mutations and, in contrast, PLA2G6-positve patients
without spheroid bodies have both been described.8
Brain or peripheral nerve pathological data for patients
with adult-onset, PLA2G6-related NBIA are not yet
available. However, a skin biopsy was normal in our
case without evidence of spheroid bodies.
Clinically, our patients showed a striking resemblance to Kufor–Rakeb syndrome11 where disease onset
was at age 12 to 15 years, within the age range of our
cases. However, none of the additional clinical features
of facial-faucial-finger mini-myoclonus, visual hallucinations, or oculogyric dystonic spasms found in further
Kufor–Rakeb syndrome cases12 were present in our
cases. There also was clinical overlap with PKAN.
However, lack of the “eye of the tiger” sign on MRI,
the absence of oromandibular dystonia, which is often
severe in PKAN patients,13 as well as the absence of
pigmentary retinopathy appears to distinguish the syndromes.14 Limb symptoms predominated in our cases;
in contrast to DYT16,3 where the craniocervical region
is prominently affected. Furthermore, reported DYT16
cases did not respond to L-dopa.
Both mutations we report herein (p.R741Q and
p.R747W) are novel; however, different mutations to
the same amino acid (R741W) have been previously described in patients with INAD.8,15 The clustering of
mutations at this part of the protein suggests this domain is critical for its function. Little, however, is
known about this function. The clinical and pathological similarity of the syndrome caused by PLA2G6 deficiency to those caused by PANK2 and ATP13A2 deficiencies suggest that all three gene products may lie on a
single biochemical pathway. The fact that we have identified gene mutations in only two of the five families
with this syndrome, and that the other three families do
not all share areas of homozygosity, suggests there must
be at least two other genes causing this syndrome. These
other genes may map to this same pathway.
This work was supported by the Intramural Research Program of
the National Institute on Aging, National Institutes of Health, Department of Health and Human Services (Intramural Program
Number Z0I AG000958-05), the Bachmann Strauss Foundation
(C.P.R., J.H.), and the Brain Research Trust, United Kingdom (JJ
Astor prize studentship, S.A.S.).
We thank Dr T. Cox for critical review of the MRI scans.
1. Zhou B, Westaway SK, Levinson B, et al. A novel pantothenate
kinase gene (PANK2) is defective in Hallervorden-Spatz syndrome. Nat Genet 2001;28:345–349.
2. Ramirez A, Heimbach A, Grundemann J, et al. Hereditary parkinsonism with dementia is caused by mutations in ATP13A2,
encoding a lysosomal type 5 P-type ATPase. Nat Genet 2006;
38:1184 –1191.
3. Camargos S, Scholz S, Simon-Sanchez J, et al. DYT16, a novel
young-onset dystonia-parkinsonism disorder: identification of a
segregating mutation in the stress response protein prkra. Lancet Neurol 2008;7:207–215.
4. Kitada T, Asakawa S, Hattori N, et al. Mutations in the parkin
gene cause autosomal recessive juvenile parkinsonism. Nature.
1998;392:605– 608.
5. Valente EM, Salvi S, Ialongo T, et al. PINK1 mutations are
associated with sporadic early-onset parkinsonism. Ann Neurol
2004;56:336 –341.
6. Bonifati V, Rizzu P, van Baren MJ, et al. Mutations in the
DJ-1 gene associated with autosomal recessive early-onset parkinsonism. Science 2003;299:256 –259.
7. Nardocci N, Zorzi G, Farina L, et al. Infantile neuroaxonal
dystrophy: clinical spectrum and diagnostic criteria. Neurology
8. Morgan NV, Westaway SK, Morton JE, et al. PLA2G6, encoding a phospholipase A2, is mutated in neurodegenerative disorders with high brain iron. Nat Genet 2006;38:752–754.
9. Khateeb S, Flusser H, Ofir R, et al. PLA2G6 mutation underlies infantile neuroaxonal dystrophy. Am J Hum Genet 2006;
10. Malik I, Turk J, Mancuso DJ, et al. Disrupted membrane homeostasis and accumulation of ubiquitinated proteins in a
mouse model of infantile neuroaxonal dystrophy caused by
PLA2G6 mutations. Am J Pathol 2008;172:406 – 416.
11. Najim al-Din AS, Wriekat A, Mubaidin A, et al. Pallidopyramidal degeneration, supranuclear upgaze paresis and
dementia: Kufor-Rakeb syndrome. Acta Neurol Scand 1994;89:
12. Williams DR, Hadeed A, al Din AS, et al. Kufor Rakeb disease:
autosomal recessive, levodopa-responsive parkinsonism with pyramidal degeneration, supranuclear gaze palsy, and dementia.
Mov Disord 2005;20:1264 –1271.
13. Schneider SA, Aggarwal A, Bhatt M, et al. Severe tongue protrusion dystonia: clinical syndromes and possible treatment.
Neurology 2006;67:940 –943.
14. Hayflick SJ, Westaway SK, Levinson B, et al. Genetic, clinical,
and radiographic delineation of Hallervorden-Spatz syndrome.
N Engl J Med 2003;348:33– 40.
15. Kurian MA, Morgan NV, MacPherson L, et al. Phenotypic
Spectrum of Neurodegeneration Associated with Mutations in
the PLA2G6gene (PLAN). Neurology (in press).
Paisan-Ruiz et al: PLA2G6-Dystonia-Parkinsonism
Без категории
Размер файла
111 Кб
locus, pla2g6, dystonic, characterization, parkinsonism
Пожаловаться на содержимое документа