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Anew locus for Parkinson's disease (PARK8) maps to chromosome 12p11.2Цq13.1

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A New Locus for
Parkinson’s Disease (PARK8) Maps to
Chromosome 12p11.2–q13.1
Manabu Funayama, MMedSc,1,4 Kazuko Hasegawa, MD, PhD,2 Hisayuki Kowa, MD, PhD,3
Masaaki Saito, MD, PhD,5 Shoji Tsuji, MD, PhD,5 and Fumiya Obata, PhD4
We performed genomewide linkage analysis of a Japanese family with autosomal dominant parkinsonism, which exhibits
clinical features compatible with those of common Parkinson’s disease. Parametric two-point linkage analysis yielded a
highest log odds (LOD) score of 4.32 at D12S345 (12p11.21). Parametric multipoint linkage analysis of the 13.6cM
interval around this marker yielded LOD scores almost uniformly of >4.0 with a Zmax of 4.71 at D12S85 (12q12).
Haplotype analysis detected two obligate recombination events at D12S1631 and D12S339 and defined the diseaseassociated haplotype in the 13.6cM interval in 12p11.2–q13.1. This haplotype was shared by all the patients and by
some unaffected carriers, suggesting that disease penetration in this family is incomplete. This low penetrance suggests
that environmental or other genetic factors modify expression of the disease. Nonparametric two-point and multipoint
linkage analyses, which are penetrance-independent, yielded Zmax LOD scores of 14.2 and 24.9 at D12S345, respectively,
strongly supporting the mapping of the parkinsonism locus in this family to 12p11.23–q13.11. This chromosome region
is different from any known locus for hereditary parkinsonism, in keeping with the unique genetic features of the
parkinsonism in this family. The nomenclature of PARK8 was assigned to the new locus.
Ann Neurol 2002;51:296 –301
DOI 10.1002/ana.10113
Parkinson’s disease (PD) (MIM 168600) is one of the
most common neurological disorders in the world, affecting ⱕ1.8% of people ⬎65 years.1 The major clinical symptoms of this disease are rigidity, bradykinesia,
tremor, and postural instability, caused by selective degeneration of dopaminergic neurons in the substantia
nigra in the midbrain. Although PD is a sporadic disease in most cases, various hereditary forms presenting
clinical phenotypes similar to those of sporadic PD
have been recognized. Molecular genetic studies on
these familial forms of parkinsonism have advanced our
understanding of the molecular mechanisms of selective degeneration of dopaminergic neurons in the midbrain substantially. To date, ␣-synuclein, parkin, tau,
and UCH-L1 have been identified as the causative
genes for familial forms of parkinsonism.2– 8 Despite
these advances, there are families with hereditary parkinsonism not linked to any of the previously identified loci.9
We previously reported a large Japanese family with
autosomal dominant parkinsonism.10,11 The clinical
features of patients in this family (Sagamihara family;
Sagamihara is the name of the area in which the family
resides) include a mean age at onset of 51 ⫾ 6 years,
presence of laterality in the parkinsonism at onset, and
favorable response to dopaminergic agents, which are
similar to those of PD. In this family, affected patients
have been observed in successive generations, men and
women are equally affected, and male to male transmission is observed, suggesting an autosomal dominant
inheritance. Neuropathological examination has been
conducted in 4 cases, which were diagnosed as “pure
nigral degeneration.” Lewy bodies were not observed.
Previous genetic studies demonstrated that Sagamihara
parkinsonism is not linked to mutations of known loci
(Hasegawa et al.12 and unpublished data). In this
study, we have undertaken genomewide linkage analysis and have identified a novel locus for familial parkinsonism on chromosome 12p11.2–q13.1.
From the Departments of 1Pediatrics, 2Internal Medicine, Kitasato
University School of Medicine; 3Kitasato University; 4Department
of Immunology, Kitasato University School of Allied Health Sciences, Sagamihara, Japan; 5Department of Neurology, Brain Research Institute, Niigata University, Niigata, Japan.
Published online Feb 21, 2002.
Received Jul 23, 2001, and in revised form Sep 21. Accepted for
publication Sep 21, 2001.
296
© 2002 Wiley-Liss, Inc.
Address correspondence to Dr Obata, Department of Immunology,
Kitasato University School of Allied Health Sciences, 1-15-1 Kitasato, Sagamihara, Kanagawa 228-8555, Japan.
E-mail: obata@ahs.kitasato-u.ac.jp
Patients and Methods
Patients and Genomic DNA
Linkage Analysis
All the patients analyzed in this study satisfied the clinical
criteria for parkinsonism, including rigidity, bradykinesia,
tremor, and postural instability. All responded well to L-dopa
treatment. No dementia or other unusual feature was observed in any of the patients. In the pedigree construction,
some familial members who had died were judged to have
had possible parkinsonism with the above symptoms on the
basis of interviews with their relatives (individuals depicted
by shadowed symbols in Fig 3). Peripheral blood was collected from 15 patients, 12 unaffected members, and 4
spouses of the Sagamihara family, with informed consent
from each subject. Genomic DNA was isolated from white
blood cells using a PUREGEN DNA isolation kit (Gentra,
MN).
Microsatellite Genotyping
First screening for the disease locus search was performed
using 382 microsatellite markers from the ABI PRISM Linkage Mapping Set Version 2 (Applied Biosystems, Foster City,
CA). Additional microsatellites for detailed screening were
chosen from the Genethon linkage map13 and the Marshfield
sex-averaged linkage map (http://research.marshfieldclinic.
org/genetics) and fluorescence labeled (6-FAM, HEX, or
NED). Polymerase chain reaction was performed using 20ng
of template DNA, 5pmol each of primers, 0.25mM each of
dATP, dTTP, dCTP, and dGTP, 50mM KCl, 15mM TrisHCl (pH 8.0), 2.5mM MgCl2, and 0.5 units of DNA polymerase (AmpliTaq Gold; Applied Biosystems). Amplification
conditions were as follows: preincubation at 95°C for 12
minutes, 10 cycles of denaturation at 94°C for 15 seconds,
annealing at 55°C for 15 seconds, and extension at 72°C for
30 seconds, 20 cycles of denaturation at 89°C for 15 seconds, annealing at 55°C for 15 seconds, and extension at
72°C for 30 seconds, and final extension for 30 minutes at
72°C. Polymerase chain reaction products were mixed with a
gel-loading cocktail containing molecular-weight standards
(GeneScan 400HDROX) and analyzed on 4% polyacrylamide denaturating gels with an ABI PRISM 377 DNA sequencer (Applied Biosystems). Microsatellite alleles were determined with GeneScan and Genotyper software.
Parametric 2-point and multipoint log odds (LOD) scores
were calculated with the MLINK and LINKMAP programs,
respectively, of the FASTLINK 4.1P package.14,15 The disease in this family was assumed to be an autosomal dominant mode of inheritance, with a gene frequency of 0.0001.
The liability classes were derived from the cumulative distribution of age of onset in 15 affected individuals: class I, ⬎71
years of age, penetrance 1.000; class II, 63 to 70 years of age,
penetrance 0.900; class III, 56 to 62 years of age, penetrance
0.700; class IV, 50 to 55 years of age, penetrance 0.500; and
class V, 0 to 49 years of age, penetrance 0.300. Allele frequencies of each microsatellite in the normal population
were determined by genotyping of 48 unrelated Japanese
individuals. The genetic distances between markers were
obtained from the Marshfield sex-averaged linkage map
(http://research.marshfieldclinic.org/genetics), estimated using Haldane’s map function. Nonparametric single-point
and multipoint linkage analysis was performed with the
GENEHUNTER program 2.0 beta r2 package.16 Because of
the memory requirements for this program (maximum bits
should be ⬍20), a limited number of family members (9
founders and 13 nonfounders) were selected for the calculation.
Results
Parametric Linkage Analysis
Allele typing of 382 microsatellite loci distributed from
chromosomes 1 to 22 was performed for 31 individuals
from four generations (15 affected, 12 unaffected, and
4 spouses). Parametric 2-point linkage analysis with the
MLINK program14 showed that D12S345 (12p11.21)
gave the highest Zmax LOD score of 4.32 (␪ ⫽ 0.02).
No other microsatellite gave LOD scores of ⬎3.0.
Thereafter, additional microsatellites around D12S345
were analyzed (Table and Fig 1). D12S1648, 0.6cM
telomeric to D12S345, gave Zmax LOD of 3.34 (␪ ⫽
0.00). Obligate recombination events were observed at
D12S1631 (12p11.23) in 2 affected individuals and at
D12S339 (12q13.11) in 1 affected individual (see lat-
Table. Parametric Two-Point Linkage LOD Scores between Autosomal Dominant Parkinsonism and Microsatellites
of Chromosome 12
Recombination fraction (␪)
Microsatellite
D12S1596
D12S1640
D12S1631
D12S87
D12S1648
D12S345
D12S1653
D12S85
D12S339
D12S1724
0.00
0.02
0.10
0.20
0.30
0.40
Zmax (␪)
⫺⬁
1.67
⫺⬁
⫺0.04
3.34
4.30
2.37
1.99
⫺⬁
⫺⬁
⫺1.03
1.58
2.16
0.06
3.33
4.32
2.28
1.91
1.24
1.02
⫺0.41
1.23
2.76
0.19
3.02
4.02
1.83
1.52
1.72
1.40
⫺0.19
0.79
2.43
0.14
2.31
3.23
1.19
0.99
1.45
1.15
⫺0.09
0.41
1.72
0.06
1.44
2.18
0.59
0.51
0.94
0.71
⫺0.04
0.13
0.84
0.02
0.59
1.00
0.16
0.16
0.40
0.29
0.00 (0.50)
1.67 (0.00)
2.76 (0.10)
0.19 (0.11)
3.34 (0.00)
4.32 (0.02)
2.37 (0.00)
1.99 (0.00)
1.72 (0.10)
1.41 (0.09)
Funayama et al: A New Parkinsonism Locus
297
defined the 13.6cM interval of the chromosome region
flanked by D12S1631 and D12S339 as the candidate
region. A haplotype within this interval was shared by
all 15 affected individuals (7 explicitly determined and
8 deduced). The haplotype, however, was also shared
by 8 unaffected individuals (5 explicitly determined
and 3 deduced). One of the carriers with the deduced
disease-associated haplotype (IV-9) died at the age of
82 with striatonigral degeneration (MIM 271930) confirmed by the neuropathological profile for typical
striatonigral degeneration having glial cytoplasmic inclusions. Among the remaining carriers of the diseaseassociated haplotype, 2 individuals were older (70 and
55 years), and 5 individuals were younger (50, 46, 33,
30, and 29 years) than the average age at disease onset
in this family (51 ⫾ 6 years). These data raise the possibility that penetrance in the Sagamihara family is incomplete.
Fig 1. Microsatellites of chromosome 12 used in this study.
The genetic distances between microsatellites were obtained
from the Marshfield sex-averaged linkage map (http://research.
marshfieldclinic.org/genetics), estimated using Haldane’s map
function. The bold bar indicates the 13.6cM interval of the
candidate region for the new parkinsonism locus.
er). To define the candidate region further, parametric
multipoint linkage analysis was performed with the
LINKMAP program15 over a 13.6cM interval of
12p11.23–q13.11, using microsatellites D12S1631,
D12S345, D12S85, and D12S339 as well as three additional microsatellites located outside this interval.
LOD scores within this 13.6cM interval were almost
uniformly of ⬎4.0 with a Zmax of 4.71 at D12S85 (Fig
2). In those affected, only analysis—both two-point
and multipoint linkage analysis—gave an even higher
Zmax LOD of 6.47 and 6.97 at D12S345, respectively.
Haplotype Analysis
Haplotype analysis encompassing the 10 microsatellites
confirmed two recombination events at D12S1631 and
D12S339 in affected individuals IV-12, V-11, and
V-7, as well as those with the deduced haplotype
(IV-10 and V-10) (Fig 3). These recombination events
298
Annals of Neurology
Vol 51
No 3
March 2002
Nonparametric Linkage Analysis
If penetrance is incomplete in the Sagamihara family,
LOD scores obtained by parametric linkage analysis
may be altered substantially by the choice of genetic
model for the analysis. We therefore performed nonparametric linkage analysis with the GENEHUNTER
program,16 in which LOD scores are calculated without prior assumptions for the genetic model. To run
the GENEHUNTER program, a limited number of
family members (9 founders and 13 nonfounders) were
used for the analysis because of the restriction in the
program (max bit ⬍20). First, 2-point linkage analysis
was performed for 56 microsatellites that displayed
LOD scores of ⬎1.0 in the parametric linkage analysis.
Again, D12S345 yielded the highest overall nonparametric LOD score, 14.2. Next, multipoint nonparaFig 2. Parametric multipoint linkage analysis. The LINKMAP program was run with the 7 microsatellites D12S1596,
D12S1640, D12S1631, D12S345, D12S85, D12S339, and
D12S1724. All 5 patients and 12 unaffected individuals were
included in the analysis, which yielded a Zmax of 4.71 at
D12S85.
Fig 3. Haplotype analysis of the Sagamihara family. Haplotypes of 31 family members were constructed based on the genotyping of
the 10 microsatellites shown in Table 1. In the cases not determined explicitly, most provable haplotypes were deduced with the
GENEHUNTER program (thin italic letters). For clarity, alleles of only the six informative microsatellites (D12S1631, D12S87,
D12S345, D12S1653, D12S85, and D12S339) are shown. In the disease-associated haplotypes, including the deduced ones
(boxed), two recombination events were observed: one at D12S1631 in affected individuals IV-10, IV-12, V-10, and V-11 and
the other at D12S339 in affected individual V-7. The 13.7cM interval region defined by these recombinations was shared by all
the patients and some unaffected carriers. Ages of the unaffected carriers were 82 (IV-9, died of STD at this age), 70 (V-18), 55
(VI-5), 50 (VI-14), 46 (V-15), 30 (VI-9), 33 (VII-4), and 29 (VII-3) years.
metric linkage analysis for the 4 microsatellites
D12S1631, D12S345, D12S85, and D12S339, the
same ones used for the parametric multipoint linkage
analysis, was performed and yielded Zmax of 24.9 at
D12S345 (Fig 4). Analysis using another set of 9
founders and 13 nonfounders demonstrated a similar
linkage profile with Zmax of 21.1 at D12S345 (data
not shown). Thus, even though the analysis included a
limited number of family members, nonparametric
linkage analysis yielded LOD scores even higher than
those obtained by parametric analysis and strongly supported the mapping of the parkinsonism locus in this
family to 12p11.23–q13.11.
Discussion
In the present study, we performed genomewide linkage analysis to localize the PD locus in the Sagamihara
family. Parametric 2-point and multipoint linkage
analysis, as well as haplotype analysis, indicated that
the disease locus in this family mapped to the 13.6cM
interval of chromosome 12p11.23–q13.11. Nonpara-
metric 2-point and multipoint linkage analysis further
confirmed this result by yielding LOD scores much
higher than those obtained by the parametric analyses.
In low-penetrance disease, as in this family, nonparametric analysis, which is penetrance-independent, is
more powerful than parametric analysis. Also, in contrast to the parametric multipoint analysis that demonstrated rather uniform LOD scores within the candidate region, the nonparametric multipoint analysis
exhibited a definitive peak at D12S345. However, we
remain unable to infer the most likely position in this
interval. Analysis of other PD families showing clinical,
pathological, and genetic features similar to those seen
in the Sagamihara family would help refine and narrow
the candidate region.
The disease-associated haplotype in this family was
shared by all of the 15 affected individuals, as well as
by 8 unaffected carriers, suggesting a low disease penetrance in this family. This low penetrance suggests
that environmental or other genetic factors modify expression of the disease. It will be particularly important
Funayama et al: A New Parkinsonism Locus
299
some 12, including many unknown genes, has been
elucidated,27 we hope that the search for the gene with
parkinsonism-causing mutations will soon be accomplished.
This study was supported by grants from the Japanese Ministry of
Education, Culture, Sports, Science and Technology (Grants-in-Aid
for Scientific Research 12031224, 12210131, and 1267061, KH)
and from Kitasato University Graduate School of Medical Sciences
(9802, FO).
References
Fig 4. Nonparametric multipoint linkage analysis. Because of
the restriction in the GENEHUNTER program (max bit
should be ⬍20), analysis was performed for 22 selected family
members. The figure shows the results obtained for 9 founders
(I-1, I-2, II-2, II-4, III-1, III-6, III-8, IV-2, IV-14) and 13
nonfounders (II-1, II-3, III-2, III-5, III-7, IV-1, IV-10, IV12, IV-13, V-5, V-7, V-17, V-19) using the 4 selected microsatellites D12S1631, D12S345, D12S85, and D12S339,
yielding a Zmax of 24.9 at D12S345. Analysis of another
combination of 9 founders (I-1, I-2, II-2, II-4, III-6, III-8,
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March 2002
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