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Molecular Findings in Familial Parkinson Disease in Spain
Janet Hoenicka, PhD;
Lídice Vidal, BS;
Blas Morales, MD, PhD;
Israel Ampuero, BS;
F. Javier Jiménez-Jiménez, MD, PhD;
Jose Berciano, MD, PhD;
Teodoro del Ser, MD, PhD;
Adriano Jiménez, MD, PhD;
Pedro G. Ruíz, MD, PhD;
Justo G. de Yébenes, MD, PhD
Arch Neurol. 2002;59:966-970.
ABSTRACT
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Background Several genetic errors in  -synuclein (Park1) and ubiquitin carboxyl-terminal–hydrolase L1(Park5) genes cause autosomal dominant familial Parkinson disease. Mutations
in the parkin gene (Park2) are the major cause of
autosomal recessive Parkinson disease.
Objective To analyze the clinical and molecular data of 19 Spanish kindreds (13
with recessive, 4 with dominant, and 2 with uncertain inheritance) who have
familial Parkinson disease.
Methods We searched for the previously described mutations in Park1 and Park5 genes and for new or described
mutations in Park2. We used single-strand conformation
polymorphism, direct sequencing, and restriction digestion of polymerase chain
reaction (PCR)–amplified genomic DNA for this study.
Results None of these families have either Park1 or Park5 mutations. We found 5 different mutations in Park2 gene in 5 of the families with recessive inheritance.
To our knowledge, 2 of these mutations, V56E and C212Y, have not been previously
reported. The other mutations found (deletion of exons 3 and 5 and 225delA)
have been described in other ethnic groups. Heterozygous carriers of a single Park2 mutation either were asymptomatic or developed clinical
symptoms in late adulthood or after brief exposure to haloperidol therapy.
Conclusions Mutations in Park2 gene account for 38% of
the families with recessive parkinsonism in Spain. We found 2 cases of simple
heterozygous Park2 mutation carriers that developed
clinical symptoms, either in late adulthood or after brief exposure to parkinsonizing
agents. Thus, hereditary Parkinson disease has more variable clinical phenotype
and molecular defects than previously thought since heterozygous mutations
could be a risk factor for parkinsonism.
INTRODUCTION
PARKINSON DISEASE (PD) (OMIM 168600) is a common neurologic disorder
of unknown origin in most cases, although several families with monogenic
inheritance have been reported.1 Two mutations
in the -synuclein gene (Park1), Ala53Thr and
Ala30Pro,2-3 and 1 mutation of
the ubiquitin carboxyl-terminal–hydrolase-L1 gene, Ile93Met,4 cause autosomal dominant
PD in a few families.
Autosomal recessive juvenile parkinsonism (ARJP) (OMIM 600116) is a
distinct clinical and genetic entity within familial PD. Autosomal recessive
juvenile parkinsonism is characterized by onset before age 40 years, good
response to levodopa therapy, prominent fluctuations, absence of Lewy bodies,
and presence of neurofibrillary degeneration in the substantia nigra.5 The gene for this disorder, mapped to chromosome 6q26-q27and
named parkin, was identified in Japanese families
with ARJP.6 Around 50% of familial ARJP and
18% cases of sporadic PD in Europe are caused by mutations in the parkin gene
(Park2) (OMIM 602544): exon deletions, exon duplications,
and point mutations.7
Parkin protein is an E3 ubiquitin-protein ligase with an ubiquitinlike
(Ubl) domain at its NH2-terminus. In its COOH-terminus there are 2 RING-finger
motifs and an IBR (in between RING fingers), organized as a RING-IBR-RING
domain.8-9 Thus, patients with
parkin mutations could have an abnormal ubiquitination pathway.
In this study, we performed the molecular analysis of 19 Spanish families
with PD. Five families with recessive inheritance have 5 different mutations
of Park2 gene, among them, V56E and C212Y. To our
knowledge, V56E and C212Y are reported for the first time. In addition, we
found 2 cases of simple heterozygous mutation in individuals that developed
clinical symptoms either in late adulthood or after a brief exposure to parkinsonizing
agents.
PATIENTS AND METHODS
CLINICAL ANALYSIS
In this study we included 19 unrelated families with PD from different
regions of Spain, 18 families of Spanish origin and 1 of Lebanese origin (family
11). Thirteen of these families have recessive inheritance (families 1-5,
8, 9, 11-15, and 18), 4 have dominant (families 6, 7, 10, and 16), and 2 have
uncertain inheritance (families 17 and 19). All patients were personally examined
by one of us (J.G.Y.) and most of them by other members of the research team.
The assignment of the clinical phenotype was performed in the Movement Disorders
Clinics of their referring hospitals or in their residences. The clinical
investigation included personal and familial clinical history, clinical examination
findings, quantitative motor examination results, and a videotaped examination.
The individuals were considered affected when they fulfilled the clinical
criteria of the slightly modified London Brain Bank.10
We required that all patients should have akinesia. Asymmetry of the initial
symptoms and good response to levodopa therapy were considered positive for
the diagnosis of PD. Informed consent was obtained from patients, members
of their families, and control subjects.
MOLECULAR ANALYSIS
High molecular weight genomic DNA, purified from peripheral blood leukocytes
according to standard methods,11 was used as
a template for polymerase chain reaction (PCR)–based methods of molecular
analysis. We looked for the previously reported missense mutations in Park1 and Park5 genes. The screening
of these mutations was performed as previously described.2-4
Restriction digestions of PCR products were done according to the manufacturer's
protocol (New England Biolabs Inc, Beverly, Mass). Plasmid DNA was used as
a positive control in -synuclein gene analysis and the restriction
maps obtained were compatible with the known sequence.
The study of Park2 gene was performed by linkage
analysis to chromosome 6q26-q27, single-strand conformation polymorphism (SSCP)
screening, and direct sequencing. Genotyping was carried out according to
standard protocols (ResGen, an Invitrogen Corp, Huntsville, Ala; also available
at: http://www.resgen.com) with the following short-tandem repeat
polymorphisms: D6S437, D6S1035, D6S1579, D6S1550, D6S305, D6S411, and D6S955.
The results were analyzed with the MLINK program of the LINKAGE package, Version
5.2 for the linkage analysis.12 The penetrance
was assumed to be 100% for a recessive model of inheritance.
The coding region of Park2 gene was amplified
using the same oligonucleotides primer pairs as previously described.6 The reactions were performed in a 25-µL reaction
mixture containing 50M potassium chloride; 10M Tris buffer, pH, 8.3; 1.75M
magnesium chloride; 25 pmol of paired primers; 10 nmol of each
deoxynucleoside triphosphate; and 1.25 U of AmpliTaq
Gold DNA polymerase (Perkin-Elmer, Applied Biosystems Division, Foster City,
Calif). Following initial denaturation at 94°C for 10 minutes, amplification
was performed through 35 cycles of 94°C for 30 seconds, 51°C for 30
seconds, and 72°C for 45 seconds, and then a final extension at 72°C
for 10 minutes.
All amplified exons were screened by SSCP analysis using the Genephor
System (Amersham Biosciences Europe GmbH, Barcelona, Spain) at 15°C according
to the manufacturer's recommendations. Patients' samples exhibiting band shifts
on SSCPs gels were subjected to direct sequence.
Families 11 and 18 were excluded from the SSCP screening because they
carried homozygous deletion of exon 3 and 5, respectively. To confirm both
deletions we designed the following inner primers: 3 inner forward 5'-AGCAG
AGCATTGTTCACATTG-3' (108 downstream from the 3 forward primer), 3 inner
reverse 5'-ACTTCCAGCTGGT GGTGAG-3' (54 nucleotides upstream of
the 3 reverse primer), 5 inner forward 5'-TCCATCTTG CTGGGATGATG-3'
(46 nucleotides downstream from the 5 forward primer), and 5 inner reverse
5'-TGCAATAAGAGGAATGAATGTG-3' (18 nucleotides upstream of the 5
reverse primer). The same primers were used for amplification and sequencing
the PCR-amplified fragments using fluorescence-based automated sequencing
technique (Applied Biosystem 373a DNA sequencer; Perkin-Elmer)
MUTATION SCREENING IN THE SPANISH POPULATION
When it was possible, the co-segregation of certain mutations with the
disease was established by restriction endonuclease digestion of PCR products
in the patient's family. The frequency of these mutations in the Spanish population
was determined in 48 individuals without neurologic diseases. Digestion conditions
were in accord with the manufacturer protocols. After digestion, fragments
were electrophoresed in 3% low–melting point agarose, stained with ethidium
bromide, and visualized under UV light.
SOUTHERN BLOT ANALYSIS
Ten micrograms of genomic DNA from patients and controls were digested
with EcoRI and PstI restriction
enzymes subjected to electrophoresis, and blotted onto a nylon membrane (Hybond
N+; Amersham Biosciences Europe GmbH) by standard procedures.11
The membrane was hybridized with parkin complementary DNA (nucleotides 290-1291)
labeled with [32P]deoxycitosine triphosphate, by use of
a Prime-It RmT Random Primer labeling kit (Stratagene, Amsterdam, the Netherlands).
RESULTS
We searched for the previously reported mutations in -synucleinand UCH-L1 genes in our families
who had PD with dominant or uncertain inheritance pattern. None of these families
carry the previously reported mutations.
We found 5 different mutations of Park2 gene
in 5 families with recessive inherited PD. The mutational spectrum includes
3 previously described deletions and 2 novel point mutations. A homozygous
deletion of exon 3 was detected in 3 patients of family 11 (Figure 1A) and the homozygous deletion of exon 5 in the available
patient of family 18 (Figure 1B).
We confirmed this finding through a second PCR with inner set of primers,
co-amplification with a control target and Southern blot analysis (Figure 1C and Figure 2). The other deletion, 255delA, present in families 3 and
13 (Figure 3), has already been
described in other European patients.7 Patients
from family 3 were homozygous for 255delA while, in family 13, the mutation
appears in only 1 allele. The other mutant allele in family 13 consists of
a heterozygous exons 8 and 9 deletion. The lesion was inferred by the alleles
pattern obtained for marker D6S411 in the individual EP13.3 and confirmed
by Southern blot (Figure 3B and Figure 2).
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Figure 1. Pedigrees of families with homozygous
deletion of 1 exon of Park2. A, Family 11. B, Family
18. C, Co-amplification of exons 3 and 5 of Park2
in affected individuals of families 18 and 11. Squares indicate men; circles,
women; solid symbols, affected individuals; open symbols, unaffected individuals;
and slashed symbols, deceased individuals. bp indicates base pair; N, normal
allele; question mark, unknown allele.
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Figure 2. Genomic Southern blot hybridization
analysis using a parkin complementary DNA fragment (nucleotides 290-1291)
as a probe. At least 9 fragments were visualized in healthy individuals (C1
and C2), as indicated by the arrows. Lost fragments are indicated by asterisks.
Patient Epe16 showed a 1,32–kilobase pair (kbp) band lost and an additional
3-kbp containing exon 6 band. A 3,49-kbp band was lost in the affected individual
EP11-4. The band pattern of the patient EP13-7 was compatible with a heterozygous
deletion of a 4,5-kbp (exon 8) and a 1,38-kbp (exon 9) bands.
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Figure 3. Pedigrees of families with 255delA
mutation. A, Family 3. B, Family 13 with genotypes for some markers. Squares
indicate men; circles, women; solid symbols, affected individuals; open symbols,
unaffected individuals; and slashed symbols, deceased individuals. bp indicates
base pair; N, normal allele; and question mark, unknown allele. Gray filled
symbol indicates drug-induced parkinsonian individual. Bars represent the
haplotypes.
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Two novel disease-causing mutations were found in 3 affected individuals
of family 4 (Figure 4A). V56E consists
in a T A transversion at position 268 in the complementary DNA that creates
a new Bst4CI restriction site. This mutation changes
the nonpolar valine residue in position 56 for the acidic polar glutamic residue
(Figure 4B). C212Y is a 736 GA
transition located in exon 6 (Figure 4C)
that replaces a highly conserved cysteine for the large aromatic amino acid
tyrosine. The 3 patients were compound heterozygotes for these 2 mutations.
The mendelian inheritance was confirmed for C212Y, which is carried by the
father. Both mutations were screened in 96 control chromosomes. Neither the
abnormal SSCP pattern corresponding to C212Y nor the new Bst4CI restriction site, created for V56E, were found in any control.
Segregation analyses showed that the 2 mutations are located in different
alleles (Figure 4A).
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Figure 4. A, Pedigree of family 4 showing
the genotype for different single-nucleotide polymorphisms and both mutations.
Markers in parentheses were inferred from other family data. Haplotype shared
by the members of each family are represented by the bars. B and C, Tracings
for exons 2 and 6 showing V56G and C212Y in heterozygous fashion. Val indicates;
Glu, glutamine; Cys, cysteine; Tyr, tyrosine; Asp, aspartic acid; and Asn,
asparagine.
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The clinical phenotype in the affected individuals from families with
known Park2 gene defects was typical of ARJP with
some exceptions. One member of family 3 (Figure 3A), a single heterozygous carrier of 255delA, developed
transient drug-induced parkinsonism at 45 years of age while being treated
with haloperidol, 2 mg/d. Another individual, a member of family 4 (Figure 4A), a single heterozygous C212Y carrier,
developed clinical symptoms at 78 years of age, while 3 of his sons with this
mutation plus V56E noted that the disease started at ages 33, 33, and 27 years.
COMMENT
We studied the 3 genes involved in familial PD. None of the studied
families have Park1 gene mutations suggesting that,
even in Mediterranean countries, mutations in the -synuclein gene are
a rare cause of PD. We also failed to find Ile93Met mutation in ubiquitin
carboxy-terminal–hydrolase L1 gene in our families.
The molecular analysis of Park2 gene allowed
the molecular characterization of 5 families, 2 with homozygous deletions
of 1 exon, 2 with a truncating mutation, and 1 with 2 combined heterozygous
novel missense mutations. The new point mutations, V56E in exon 2 and C212Y
in exon 6, lie in different parkin functional domains. The nonconservative
V56E amino acid change, in the Ubl domain of parkin, is located in a conserved
position among ubiquitins from phylogenetically distanced organisms Rattus norvegicus (swall: Q9JM64), Mus
musculus (swall: AA613892), Drosophila melanogaster (swall: Q9VP72), and Tetrahymena pyriformis
(swall: Q27194)). Since the Ubl domain is required for the recognition of
the ubiquitinated proteins, ParkinV56E could be unable to bind
with its target protein, which might result in pathological accumulation of
the target and negative effects on the central nervous system.
C212Y is located in a highly conserved residue and very close to the
first RING-finger motif that is required for E3 ubiquitin-protein ligase activity
of parkin. This region contains up to 8 conserved cysteine residues that may
be involved in disulphide bridges and, therefore, a cysteine substitution
could change the secondary structure of the protein besides the first RING
finger.
Regarding the 255delA mutation, present in families 3 and 13, we found
by the analysis of microsatellites that the deletion lies in 3 different haplotypes
(Figure 3). The sequence analysis
of the region around 255delA showed the presence of short direct repeats at
the 3' region, suggesting that a recurrence mechanism is involved.
Three patients with early-onset PD from family 4 are combined heterozygous
for V56E and C212Y. One brother, 45 years old, a heterozygous carrier of the
mutation V56E, is asymptomatic. The father, however, a single heterozygous
carrier for C212Y mutation, developed parkinsonism at the age of 78 years.
Recently, Klein et al13 identified, in a large
family, heterozygous patients with Park2 mutations
in 1 allele clinically indistinguishable from most patients with idiopathic
PD. Moreover, positron emission tomographic analysis in this family indicates
a preclinical disease process in the asymptomatic heterozygous carrier subjects.14 Our data agree with those findings and expand our
knowledge about the influence of Parkin abnormalities in PD.
In addition, we have evidence that heterozygous mutations in Park2 could be a risk factor for drug-induced parkinsonism. Eight members
of family 3, heterozygous carriers of the 255delA mutation, are asymptomatic,
but 1 of the 8 developed parkinsonism when treated with haloperidol, 2 mg/d.
This patient fully recovered after discontinuation of the drug. Neuroleptic-induced
parkinsonism is more frequent in relatives of patients with PD15
and that increased risk has never been explained. Thus, it is conceivable
that individuals with half activity of E3 ubiquitin-ligase parkin as heterozygous
carriers could be more susceptible to drug-induced parkinsonism, to the effects
of aging, or to environmental agents. These findings, which could be overlooked
as merely anecdotical, need to be investigated in a larger and specifically
designed study. The role of parkin in sporadic PD remains to be discovered,
but our observations suggest that Park2 might be
implied in the origin of the more frequent late-onset PD and drug-induced
parkinsonism.
The study of whether the heterozygous state predisposes to disease will
help explain parkin implications, if any, in common sporadic PD and drug-induced
parkinsonism either in late adulthood or after a brief exposure to parkinsonizing
agents.
AUTHOR INFORMATION
Accepted for publication February 1, 2002.
Author contributions: Study concept and design (Drs Hoenicka, Morales, and de Yébenes); acquisition
of data (Drs Hoenicka, Jiménez-Jiménez, Berciano,
del Ser, Jiménez, Ruíz, and de Yébenes and Ms Vidal); analysis and interpretation of data (Drs Hoenicka
and de Yébenes, Ms Vidal, and Mr Ampuero); drafting of the manuscript (Drs Hoenicka and Berciano and Ms Vidal); critical revision
of the manuscript for important intellectual content (Drs
Hoenicka, Morales, Jiménez-Jiménez, Berciano, del Ser, Jiménez,
Ruíz, and de Yébenes, Ms Vidal, and Mr Ampuero); statistical
expertise (Dr Jiménez); obtained funding (Drs Morales, Ruíz, and de Yébenes); administrative,
technical, and material support (Dr del Ser); study
supervision (Drs Hoenicka, Berciano, and de Yébenes).
This work was supported by Dirección General de Investigación
de la Comunidad de Madrid, Madrid; the Fundación Caja Rural de Soria,
Soria, Spain; the Fundación Ramón Areces, Madrid; and the Fondo
de Investigaciones Sanitarias, Madrid.
We are grateful to the patients and family members for participating
in the study and for donating blood samples for DNA extraction.
Corresponding author: Janet Hoenicka, PhD, Banco de Tejidos para
Investigaciones Neurológicas, Facultad de Medicina, Universidad Complutense
de Madrid, Pabellón III, sótano, Avda Complutense s/n Madrid
28040, Spain (e-mail: jhoenicka{at}cbm.uam.es).
From the Unidad de Diagnóstico Molecular, Banco de Tejidos para
Investigaciones Neurológicas, Madrid (Drs Hoenicka and de Yébenes,
Ms Vidal, and Mr Ampuero), Servicios de Neurología, Hospital Universitario
S Cecilio, Granada (Dr Morales), Hospital Príncipe de Asturias, Alcalá
de Henares (Dr Jiménez-Jiménez), Hospital Severo Ochoa, Leganés
(Dr del Ser), Hospital Ramón y Cajal, Madrid, and Hospital Marqués
de Valdecilla, Cantabria (Dr Jiménez), and Fundación Jiménez
Díaz, Madrid (Drs Berciano, Ruíz, and de Yébenes), Spain.
REFERENCES
| |
1. Hoenicka J, Varo Acosta J. Cuánto de hereditario y cuánto de ambiental hay en la
enfermedad de Parkinson. Continua Neurológica. 2000;3:1-12.
2. Polymeropoulos MH, Lavedan C, Leroy E, et al. Mutation in the -synuclein gene identified in families with
Parkinson's disease. Science. 1997;276:2045-2047.
FREE FULL TEXT
3. Krüger R, Kuhn W, Müller T, et al. ALA30PRO mutation in the gene encoding -synuclein in Parkinson's
disease. Nat Genet. 1998;18:106-108.
FULL TEXT
|
ISI
| PUBMED
4. Leroy E, Boyer R, Auburger G, et al. The ubiquitin pathway in Parkinson's disease. Nature. 1998;395:451-452.
FULL TEXT
| PUBMED
5. Mori H, Kondo T, Yokochi M, et al. Pathologic and biochemical studies of juvenile parkinsonism linked
to chromosome 6q. Neurology. 1998;51:890-892.
FREE FULL TEXT
6. Kitada T, Asakawa S, Hattori N, et al. Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature. 1998;392:605-608.
FULL TEXT
| PUBMED
7. Abbas N, Lucking CB, Ricard S, et al for the French Parkinson's Disease Genetics Study Group and the European
Consortium on Genetic Susceptibility in Parkinson's Disease. A wide variety of mutations in the parkin gene are responsible for
autosomal recessive parkinsonism in Europe. Hum Mol Genet. 1999;8:567-574.
FREE FULL TEXT
8. Shimura H, Hattori N, Kubo S, et al. Familial Parkinson disease gene product, parkin, is a ubiquitin-protein
ligase. Nat Genet. 2000;25:302-305.
FULL TEXT
|
ISI
| PUBMED
9. Imai Y, Soda M, Takahashi R. Parkin suppresses unfolded protein stress-induced cell death through
its E3 ubiquitin-protein ligase activity. J Biol Chem. 2000;275:35661-35664.
FREE FULL TEXT
10. Hughes AJ, Daniel SE, Blankson S, Lees AJ. A clinicopathologic study of 100 cases of Parkinson's disease. Arch Neurol. 1993;50:140-148.
ABSTRACT
11. Sambrook J, Maniatis T, Fritsch EF. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press;
1989.
12. Lathrop GM, Lalouel JM, Julier C, Ott J. Strategies for multilocus linkage analysis in humans. Proc Natl Acad Sci U S A. 1984;81:3443-3446.
FREE FULL TEXT
13. Klein C, Pramstaller PP, Kis B, et al. Parkin deletions in a family with adult-onset, tremor-dominant parkinsonism:
expanding the phenotype. Ann Neurol. 2000;48:65-71.
FULL TEXT
|
ISI
| PUBMED
14. Hilker R, Klein C, Ghaemi M, et al. Positron emission tomographic analysis of the nigrostriatal dopaminergic
system in familial parkinsonism associated with mutations in the parkin gene. Ann Neurol. 2001;49:367-376.
FULL TEXT
|
ISI
| PUBMED
15. Myrianthopoulos NC, Waldrop FN, Vincent BL. A repeat study of hereditary predisposition in drug induced parkinsonism. In: Barbeau A, Brunette JR, eds. Progress in Neurogenetics. Amsterdam, the Netherlands: Excerpta Medica; 1967;175:486-491.
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