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DJ-1 mutations and parkinsonism-dementia-amyotrophic lateral sclerosis complex.

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(RanGEF), the Db1 and pleckstrin homology (DH/PH)
domains (RhoGEF), the two MORN (membrane occupation and recognition nexus) repeat motifs, and the
VPS9 (vacuolar protein sorting 9) homology domain
(GEF for GTPase Rab5), which functions as an essential
element for the ALSIN-associated Rab5GEF activity.
There is now evidence that the absence of a functional
VPS9 domain of ALSIN is sufficient to cause neurodegeneration.8 –11
In conclusion, we present an ALS2 patient with a
novel mutation in the ALS2 gene, showing an earlier
age of onset than previously described cases, that is, in
his second year in contrast with published ALS2 patients (6.5 ⫾ 2.3 years in the patients described by Ben
Hamida and colleagues5 or 3–23 years as reported by
Ben Hamida and colleagues12). Furthermore, the progression rate was more rapid, leaving the patient
wheelchair-bound and anarthric at age 18 years in contrast with the reported patients who were wheelchairbound between the 40th and 50th year. Additional genetic and nongenetic factors may play an essential role
in the modification of the phenotype.
12. Ben Hamida B, Hentati F, Ben Hamida C. Hereditary motor
system diseases. Brain 1990;113:347–363.
13. Antonarakis SE. Recommendations for a nomenclature system
for human gene mutations. Nomenclature Working Group.
Hum Mutat 1998;11:1–3.
DJ-1 Mutations and
Parkinsonism-DementiaAmyotrophic Lateral
Sclerosis Complex
Grazia Annesi, PhD,1 Giovanni Savettieri, MD,2
Pierfrancesco Pugliese, MD,3 Marco D’Amelio, MD,2
Patrizia Tarantino PhD,1 Paolo Ragonese, MD,2
Vincenzo La Bella, MD,2 Tommaso Piccoli, MD,2
Donatella Civitelli, PhD,1 Ferdinanda Annesi, PhD,1
Brigida Fierro, MD,2 Federico Piccoli, MD,2
Gennarina Arabia, MD,3 Manuela Caracciolo, MD,1
Innocenza Claudia Cirò Candiano, PhD,1 and
Aldo Quattrone, MD1,3
1. Yang Y, Hentati A, Deng HX, et al. The gene encoding alsin,
a protein with three guanine-nucleotide exchange factor domains, is mutated in a form of recessive amyotrophic lateral
sclerosis. Nat Genet 2001;29:160 –165.
2. Eymard-Pierre E, Lesca G, Dollet S, et al. Infantile-onset ascending hereditary spastic paralysis is associated with mutations
in the alsin gene. Am J Hum Genet 2002;71:518 –527.
3. Shaw PJ. Genetic inroads in familial ALS. Nat Genet 2001;29:
4. Hadano S, Hand CK, Osuga H, et al. A gene encoding a putative GTPase regulator is mutated in familial amyotrophic lateral sclerosis 2. Nat Genet 2001;29:166 –173.
5. Hentati A, Bejaoui K, Pericak-Vance MA, et al. Linkage of recessive familial amyotrophic lateral sclerosis to chromosome
2q33–q35. Nat Genet 1994;7:425– 428.
6. Hentati A, Ouahchi K, Pericak-Vance MA, et al. Linkage of a
commoner form of recessive amyotrophic lateral sclerosis to chromosome 15q15–q22 markers. Neurogenetics 1998;2:55– 60.
7. Hand CK, Devon RS, Gros-Louis F, et al. Mutation screening
of the ALS2 gene in sporadic and familial amyothrophic lateral
sclerosis. Arch Neurol 2003;60:1768 –1771.
8. Otomo A, Hadano S, Okada T, et al. ALS2, a novel guanine
nucleotide exchange factor for the small GTPase Rab5, is implicated in endosomal dynamics. Hum Mol Genet 2003;12:
9. Topp JD, Gray NW, Gerard RD, et al. Alsin is a Rab5 and
Rac1 guanine nucleotide exchange factor. J Biol Chem 2004;
10. Kunita R, Otomo A, Mizumura H, et al. Homooligomerization of ALS2 through its unique carboxyl-terminal
regions is essential for the ALS-associated Rab5 guanine nucleotide exchange activity and its regulatory function on endosome
trafficking. J Biol Chem 2004;279:38626 –38635.
11. Kanekura K, Hashimoto Y, Kita Y, et al. A Rac1/
phosphatidylinositol 3-kinase/Akt3 anti-apoptotic pathway,
triggered by alsinLF, the product of the ALS2 gene, antagonizes
Cu/Zn-superoxide dismutase (SOD1) mutant-induced motoneural cell death. J Biol Chem 2005;280:4532– 4543.
DJ-1 gene mutations have been found to cause early-onset
Parkinson’s disease. We report a family from southern Italy with three brothers affected by early-onset parkinsonism, dementia, and amyotrophic lateral sclerosis. Molecular analysis of the DJ-1 gene in two living patients showed
a novel homozygous mutation in exon 7 (E163K) and a
new homozygous mutation (g.168_185dup) in the promoter region of the gene. Both mutations cosegregated
with the disease and were detected in a heterozygous state
in the patients’ mother and their healthy siblings. Our
findings expand the spectrum of clinical presentations associated with mutations in DJ-1 gene.
Ann Neurol 2005;58:803– 807
Mutations in DJ-1 gene have been recently shown to
cause autosomal recessive early-onset Parkinson’s disease (EOPD) in a large Dutch family and in a small
consanguineous Italian family.1 Subsequent to this ini-
From the 1Institute of Neurological Sciences, National Research
Council, Piano Lago di Mangone, Cosenza; 2Department of Neurology, Ophthalmology, Otorinolaringology and Psychiatry, University of Palermo, Palermo; and 3Istitute of Neurology, University
Magna Graecia, Catanzaro, Italy.
Received Jun 24, 2005, and in revised form Aug 22. Accepted for
publication Aug 24, 2005.
Published online Oct 24, 2005, in Wiley InterScience
( DOI: 10.1002/ana.20666
Address correspondence to Dr Quattrone, Cattedra e U.O. di Neurologia, Università Magna Graecia, via T. Campanella, 88100 Catanzaro, Italy. E-mail:
© 2005 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
tial finding, several additional DJ-1 mutations were
identified in subjects with EOPD.2– 6 We describe a
family from southern Italy with three brothers affected
by a complex disorder characterized by early-onset
parkinsonism-dementia-amyotrophic lateral sclerosis
(EOPD-D-ALS). The analysis of the DJ-1 gene
showed a novel homozygous mutation (E163K) in
exon 7 and a novel homozygous mutation
(g.168_185dup) in the promoter region of this gene in
living affected subjects.
Subjects and Methods
Informed written consent was obtained from five individuals
of our study family. The family pedigree is shown in Figure
1. The patients (III-2, III-3, and III-4) showed clinical pictures characterized by EOPD-D-ALS (definite ALS, III-2;
probable ALS [laboratory supported], III-3, III-4, according
to El Escorial revised criteria7). Parents of these three patients were second cousins; the father (II-7) died when he
was 77 years old from a pneumonia infection. He did not
suffer from neurological diseases. On February 2004, we visited the patients’ mother (II-8; 1925) and their siblings
(III-1, 1952; and III-5, 1954) who showed neither signs nor
symptoms of neurological disorders.
Patient 1 (III-2: 1951), at age 36, reported progressive
weakness of the upper and lower limbs. At the age 37 years,
neurological examination was characterized by muscle atrophy of the upper and the lower extremities with diffuse fasciculation. Needle electromyography (EMG) showed a diffuse denervation. Cerebrospinal fluid (CSF) examination
showed no abnormalities. There was marked cognitive impairment (Wechsler Adult Intelligence Scale score, 59). Approximately 30 months later, he reported rigidity, resting
tremor, and bradykinesia. At the age of 40 years, he had
increased salivation, progressive dysphagia, and severe weight
loss with marked wasting of muscles. His speech became dysartric. Neurological examination showed signs of involvement of upper and lower motor neuron (severe bulbar involvement, muscle atrophy and brisk reflexes at four limbs,
bilateral Babinski’s sign) and parkinsonism. He died when he
was 43 years old from respiratory failure.
Patient 2 (III-3: 1960), at age 35 years, reported loss of
initiative and disinterest in daily activities. At age 39 years,
neurological examination showed mild parkinsonism, dysartria, brisk reflexes, right Babinski’s sign, moderate atrophy of
the distal lower limbs, and a mild intellectual impairment
(Mini-Mental State Examination [MMSE] score, 22). In addition, an abnormal eating behavior (bulimia) was reported
by the patient’s relatives. EMG showed a diffuse denervation,
whereas motor and sensory evoked potentials and CSF were
normal. A r-CBF SPECT showed bilateral decreased perfusion of basal ganglia and the frontal and parietal lobes. In
February 2004, neurological examination was mainly characterized by severe parkinsonism with postural instability,
slurred speech, occasional dysphagia, tongue fasciculation,
urinary incontinence, and difficulty walking. Deep reflexes
were brisk and Babinski’s sign was present bilaterally. Muscle
atrophy was prevalent at the lower limbs with diffuse fasciculation. A further progression of cognitive impairment
(MMSE score, 13) was also observed. On EMG examination, fibrillation potential and fasciculation were diffusely recorded from the muscles of the lower and upper limbs,
whereas motor and sensory conduction velocities were within
normal limits. Finally, the feeding misconduct increased over
time, and the patient also developed an aggressive behavior.
Patient 3 (III-4;1969) was healthy until the age of 24
years when he reported slowness and mild difficulty in speaking. A neurological examination, performed 2 years later,
showed parkinsonism, dysartria, and brisk reflexes of the
lower limbs. The MMSE score was 25. EMG demonstrated
diffuse denervation with normal motor and sensory conduction velocities; motor and sensory evoked potentials and CSF
were normal. Cerebral magnetic resonance imaging showed
mild cortical atrophy. The patient was treated with L-dopa
with mild to moderate benefit, but he reported dyskinesias
and hallucinations within a few months. In February 2004,
clinical examination showed the progression of the parkinsonian syndrome and the appearance of limited voluntary upward eye movement. In addition, brisk reflexes, bilateral
Babinski’s sign, dysphagia, dysartria, and urinary incontinence were present. A marked decline of cognitive function
was also observed (MMSE score, 10). The EMG examination showed a pattern similar to that described for Patient 2.
On the occasion of his last visit, the patient’s relatives reported that he also suffered from bulimia and that in the last
few years he had developed an antisocial behavior mainly
characterized by swearing and shouting in public.
Fig 1. Pedigree of the Italian family with double homozygous mutation in the DJ-1 gene. Squares represent men, and circles represent women. Filled symbols are clinically affected individuals; half-filled symbols are healthy carriers. Slashed symbols represent deceased individuals. Black dots indicate obligate carriers. (⫹/⫹ ⫽ double homozygous mutation; ⫹/⫺ ⫽ double heterozygous mutation; nd ⫽ not determined)
Annals of Neurology
Vol 58
No 5
November 2005
Fig 2. (A) Sequencing from genomic DNA showing the E163K mutation in our study family. The arrow indicates the site of the
substitution. (B) The mutation was verified by the loss of a TaqI restriction enzyme site. Marker DNA (M), PCR product undigested (lane 1), heterozygous state (Subject II-8) producing three fragments of 504, 383, and 121bp (lane 2), homozygous state
(Patients III-3 and III-4) showing only an uncleaved band of 504bp (lanes 3 and 4), digestion of normal alleles producing two
fragments of 383 and 121bp (lane 5).
Genetic Analysis
We analyzed five individuals from our study family: II-8,
III-1, III-3, III-4, and III-5. Moreover, we also analyzed 700
healthy controls from southern Italy. Polymerase chain reaction (PCR) amplification of Parkin, PINK1, MAP␶, SOD-1,
ALS2, and DJ-1 exons was performed on all DNA samples.
Bidirectional dideoxy chain terminator sequencing was performed according to the manufacturer’s instructions (BigDye; Applied Biosystems, Foster City, CA) and the products
were electrophoresed on the ABI373 DNA sequencer (Applied Biosystems).
Gene Dosage studies of all 12 parkin exons were performed using real-time PCR 7900 HT-SDS (Perkin-Elmer
Applied Biosystems, Foster City, CA).
No mutations were detected in the Parkin, PINK1, MAP␶,
SOD-1 and ALS2 genes. Direct sequencing of the DJ-1 gene
displayed a G-A novel homozygous missense mutation at position 3385 (Gene Bank Accession Number ALO34417) in
exon 7 in the two affected brothers resulting in the substitution of a highly conserved E (glutamic acid) at position
163 of the DJ-1 protein by a K (lysine) (Fig 2A). This mutation was confirmed by digestion of the restriction enzyme
TaqI (see Fig 2B) and fully cosegregated with the disease in
our study family. This mutation was not found in 500 chromosomes of the control subjects from southern Italy. The
mother (II-8) and the healthy siblings (III-1, III-5) of the
patients were heterozygous for the E163K mutation. Moreover, we identified a homozygous mutation (g.168_185dup)
in the promoter of the DJ-1 gene (Fig 3A, B) of the patients.
This mutation, located at the level of the polymorphism
(g.168_185del) previously described in the promoter of the
DJ-1 gene,8 also showed complete cosegregation with the
disease and was found in a heterozygous state in the mother
and in the healthy siblings (III-1, III-5) of the patients. The
duplication was not found in 1,400 chromosomes from our
control population.
In this study, we found a double homozygous mutation in the DJ-1 gene in a family from southern Italy
with a complex phenotype characterized by EOPD-DALS.
One of these mutations was a missense mutation in
exon 7 of the DJ-1 gene that resulted in an amino acid
substitution of lysine with glutamic acid (E163K), a
residue highly conserved throughout evolution,
whereas the other one was a new mutation
(g.168_185dup) located in the promoter region of
DJ-1 gene. The latter is a potentially functional mutation as well, because it is localized 168bp after the transcriptional activation site and approximately 50bp
downstream of an SP1 binding site9 and could potentially influence the level of DJ-1 promoter transactivation or the transcript stability and translation efficiency, thus contributing to the complexity of the
Mutations in DJ-1 lead to an early onset form of
parkinsonism with a slow disease progression and a
good response to L-dopa.1 The clinical picture of our
patients was strongly different from that described in
DJ-1 related disease. Indeed, it was characterized by
EOPD-D-ALS. Speech difficulties were the more fre-
Annesi et al: DJ-1 and ALS
Fig 3. (A) Agarose gel (3%) stained with ethidium bromide illustrating the genotyping for the g.168_185del polymorphism and the
g.168_185dup mutation. (B) Representative sequence identifying the g.168_185dup mutation. I: homozygote del/del; II: homozygote
ins/ins; III: heterozygote ins/del; IV: homozygote dup/dup; V: heterozygote ins/dup.
quent presenting symptom, whereas parkinsonism, cognitive decline, pyramidalism, and amyotrophy were the
forefront of the developed clinical picture in all the patients. However, variability of the clinical picture between the three affected brothers was observed in the
course of the disease. In Patient III-2, a severe ALS was
the prominent clinical feature, whereas parkinsonism
and cognitive impairment were the prominent clinical
findings in the other two patients (III-3, III-4) who
also showed behavioral abnormalities characterized by
aggressive, antisocial behavior, and feeding misconduct.
Parkinsonism, dementia, and ALS may develop together in the same patient. A relationship among these
disorders was first noted in Guam10 where a variant of
ALS and a parkinsonism dementia complex was described in the Chamorro people. Both disorders, which
manifest together in approximately 5% of cases, have
overlapping neuropathological findings, with neurofibrillary tangles containing tau proteins in the spinal
cord and brain,11 but no mutations in the MAP␶
gene.12 More recently, an association between familial
frontal-temporal dementia with parkinsonism (FTDP)
and ALS, with or without MAP␶ mutations, has been
reported.13–16 All the reported families have abundant
Annals of Neurology
Vol 58
No 5
November 2005
tau-positive inclusions in the brain, suggesting that tau
pathology could be a central pathological event in this
degenerative disease.17
The mechanism by which DJ mutations can result
in a complex clinical phenotype characterized by
EOPD-D-ALS is unknown. Possible roles for DJ-1 as a
sensor of oxidative stress or a molecular chaperone are
currently being discussed.18 Because oxidative stress
may be involved in the pathogenesis of some neurodegenerative disorders such as PD and ALS, it can be
hypothesized that DJ-1 knockdown by enhancing the
susceptibility to oxidative stress could contribute to the
development of these motor disorders. On the other
hand, DJ-1 could have a chaperone activity protecting
cells from abnormally aggregated proteins.18 A recent
study19 showed that the DJ-1 protein was expressed
and colocalized within a subset of pathological tau inclusions typically occurring in several tauopathies, including FTDP-17, and that solubility of the DJ-1 protein was altered in association with its aggregation
within these inclusions, suggesting that the sequestration of the DJ-1 protein within the inclusions could
decrease its chaperone activity for tau and other proteins.
Although our study family lacks neuropathological
data, the striking similarity between the complex clinical phenotype described here and that observed in
some tauopathies, such as FTDP-17 with ALS, further
supports the hypothesis of a link between DJ-1 and tau
protein in the pathogenesis of some neurodegenerative
17. Spillantini MG, Goedert M. Tau protein pathology in neurodegenerative diseases. Trends Neurosci 1998;21:428 – 433.
18. Bonifati V, Oostra BA, Heutink P. Linking DJ-1 to neurodegeneration offers novel insights for understanding the pathogenesis of Parkinson’s disease. J Mol Med 2004;82:163–174.
19. Rizzu P, Hinkle DA, Zhukareva V, et al. DJ-1 colocalizes with
tau inclusions: a link between parkinsonism and dementia. Ann
Neurol 2004;55:113–118.
1. Bonifati V, Rizzu P, Van Baren MJ, et al. Mutations in the
DJ-1 gene associated with autosomal recessive early-onset parkinsonism. Science 2003;229:256 –259.
2. Abou-Sleiman PM, Healy DG, Quinn N, et al. The role of
pathogenic DJ-1 mutations in Parkinson’s disease. Ann Neurol
3. Hague S, Rogaeva E, Hernandez D, et al. Early-onset Parkinson’s disease caused by a compound heterozygous DJ-1 mutation. Ann Neurol 2003;54:271–274.
4. Hering R, Strauss KM, Tao X, et al. Novel homozygous
p.E64D mutation in DJ1 in early onset Parkinson disease
(PARK7). Hum Mutat 2004;24:321–329.
5. Hedrich K, Djarmati A, Schafer N, et al. DJ-1 (PARK 7) mutations are less frequent than Parkin (PARK2) mutations in
early-onset Parkinson disease. Neurology 2004;62:389 –394.
6. Djarmati A, Hedrich K, Svetel M, et al. Detection of Parkin
(PARK2) and DJ-1 (PARK7) mutations in early-onset Parkinson disease: Parkin mutation frequency depends on ethnic origin of patients. Hum Mutat 2004;23:525.
7. Brooks BR, Miller RG, Swash M, et al. for the World Federation of neurology Group on Motor Neuron Diseases. El Escorial revisited: revised criteria for the diagnosis of amyotrophic
lateral sclerosis. Amyotroph Lateral Scler Other Motor Neuron
Disord 2000;1:293–299.
8. Eerola J, Hernandez D, Launes J, et al. Assessment of a DJ-1
(PARK7) polymorphism in Finnish PD. Neurology 2003;61:
1000 –1002.
9. Taira T, Takahashi K, Kitagawa R, et al. Molecular cloning of
human and mouse DJ-1 genes and identification of Sp1dependent activation of the human DJ-1 promoter. Gene 2001;
10. Arnold A, Edgren DG, Palladino VS. Amyotrophic lateral
sclerosis: fifty cases observed in Guam. J Nerv Ment Dis 1953;
11. Buee-Scherrer V, Buee L, Hof PR, et al. Neurofibrillary degeneration in amyotrophic lateral sclerosis/parkinsonism-dementia
complex of Guam. Immunochemical characterization of tau
proteins. Am J Pathol 1995;146:924 –932.
12. Poorkaj P, Tsuang D, Wijsman E, et al. TAU as a susceptibility
gene for amyotrophic lateral sclerosis-parkinsonism dementia
complex of Guam. Arch Neurol 2001;58:1871–1878.
13. Wilhelmsen KC, Lynch T, Pavlou E, et al. Localization of the
disinhibition-dementia-parkinsonism-amyotrophy complex to
17q21–22. Am J Hum Genet 1994;55:1159 –1165.
14. Clark LN, Poorkaj P, Wszolek Z, et al. Pathogenic implications
of mutations in the tau gene in pallido-ponto-nigral degeneration and related neurodegenerative disorders linked to chromosome 17. Proc Natl Acad Sci USA 1998;95:13103–13107.
15. Zarranz JJ, Ferrer I, Lezcano E, et al. A novel mutation
(K317M) in the MAPT gene causes FTDP and motor neuron
disease. Neurology 2005;64:1578 –1585.
16. Wilhelmsen KC, Forman MS, Rosen HJ, et al. 17q-linked
frontotemporal dementia-amyotrophic lateral sclerosis without
tau mutations with tau and ␣-synuclein inclusions. Arch Neurol 2004;61:398 – 406.
The Role of the ND5 Gene
in LHON: Characterization
of a New, Heteroplasmic
LHON Mutation
Vladimir Mayorov, PhD,1 Valerie Biousse, MD,2,3
Nancy J. Newman, MD,2– 4 and Michael D. Brown, PhD1
Leber’s hereditary optic neuropathy (LHON) causes central vision loss from bilateral optic neuropathy. Although
13 mitochondrial DNA (mtDNA) mutations are strongly
associated with LHON, only three account for roughly
90% of cases and thus are found in multiple independent
LHON families. The remaining LHON mutations are
rare. Here, we describe the clinical and genetic characterization of a new LHON mtDNA mutation. The 12848T
mutation alters a highly conserved amino acid in the
ND5 complex I gene, is not found in controls, and is
heteroplasmic. Despite ND5 being the largest of the
mtDNA complex I genes, ND5 mutations are quite rare
in LHON.
Ann Neurol 2005;58:807– 811
Mitochondrial DNA (mtDNA) mutations cause Leber’s hereditary optic neuropathy (LHON), a form of
blindness caused by retinal ganglion cell atrophy. To
date, 40 different mutations have been associated
with LHON.1,2 Of these, 10 (3460A, 3733A, 4171A,
10663C, 11778A, 14482A/G, 14484C, 14495G, and
14568T) variants represent confirmed “primary”
From the 1Division of Basic Medical Sciences, Mercer University
School of Medicine, Macon; and Departments of 2Ophthalmology,
Neurology, and 4Neurological Surgery, Emory University School of
Medicine, Atlanta, GA.
Received May 23, 2005, and in revised form Jul 11. Accepted for
publication Aug 17, 2005.
Published online Oct 24, 2005, in Wiley InterScience
( DOI: 10.1002/ana.20669
Address correspondence to Dr Brown, Mercer University School of
Medicine, Macon, GA 31207. E-mail:
© 2005 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
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complex, mutation, dementia, lateral, sclerosis, amyotrophic, parkinsonism
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