Parkinsonism and Related Disorders 14 (2008) 520e522
www.elsevier.com/locate/parkreldis
Letter to the Editor
Investigation of genes related to familial forms of Parkinson’s
disease e With focus on the Parkin gene
1. Introduction
Of the recessively inherited forms of Parkinson’s disease
(PD), Parkin (PARK2) associated PD appears to be the most
frequent. Mutations in the Parkin gene, were initially reported
to cause autosomal recessive juvenile onset (before 20 years)
PD, however, Parkin mutations have later been found in cases
of early-onset PD (below 40e50 years) [1]. Mutations linked
to recessive forms of PD have also been found in the genes
DJ-1 and PINK1 [1]. Genes causing dominantly inherited
PD are a-Synuclein (PARK1) and LRRK2 (PARK 8), where
LRRK2 mutations are the most common genetic cause of
familial and sporadic PD detected so far [1]. Another gene
implicated in PD is UCHL-1, but the data about its role in
the aetiology of the disease are equivocal [1].
Our Swedish PD population contains a subgroup of early-
onset patients (50 years) and to gain insight into the role of
the Parkin gene in the disease among these patients we screened
the coding parts of the gene. We were further encouraged by the
fact that no earlier studies of this gene have been performed in
a Swedish PD population. By using Multiplex Ligation-
dependent Probe Amplification (MLPA) to detect potential
exon rearrangements in the Parkin gene it was also possible,
from the same probemix, to get information on aberrant copy
numbers of parts of the genes a-synuclein, UCHL-1, DJ-1,
PINK1 and PACRG (which means Parkin co-regulated gene
because it shares promoter region with the Parkin gene [2]).
2. Subjects and methods
2.1. Subjects
Sixty-three patients with early-onset PD (50 years) were chosen from
a large PD-material, consisting of patients recruited from the Swedish cities
Stockholm (n ¼ 173), Göteborg (n ¼ 116), Skövde (n ¼ 12) and Falköping
(n ¼ 45), for direct sequencing of the Parkin gene and MLPA analysis. Sixteen
of them reported a family history of the disease. Only the MLPA analysis was
also performed on 10 additional patients with a family history of the disease,
but with a later age of onset. All patients fulfilled the PDS Brain Bank criteria
for idiopathic PD [3], except that some cases had more than one affected
relative. The mean age of onset in the group of patients with an age of onset
at or before 50 years was 44 years. The controls used in the MLPA analysis,
were individuals visiting hospitals and care centres in Göteborg. All subjects
in the study had provided informed consent, and the study was approved by
the ethical committees at Göteborg University and Karolinska Institutet.
1353-8020/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.parkreldis.2007.10.013
2.2. Direct sequencing
Direct sequencing of the coding parts of the Parkin gene was performed
using the BigDye terminator chemistry (Applied Biosystems, Foster City,
CA, USA). PCR products were loaded on an ABI 3730 capillary DNA
sequencer and analysed with the SeqScape software version 2.5 (Applied
Biosystems, Foster City, CA, USA).
2.3. MLPA
MLPA analysis was carried out according to the manufacturer’s recom-
mendations (MRC Holland, Amsterdam, The Netherlands). For the genes of
interest in the present study, the P051 mix contained probes for a-synuclein
exons 1, 4, 5, 6 and the A30P mutation, Parkin exons 1e12, DJ-1 exons 1,
3, 5, 7 and PINK1 exons 1e8. The P052 mix contained probes for a-synuclein
exon 2, UCHL-1 exons 1,4,5,9, Parkin exons 2, 4e10, 12, and PACRG exon 1.
In the latest version of the P051/P052 mixes (lot 0505), additional LRRK2-
probes had been included. The amplification products were separated by elec-
trophoresis on an ABI 3730 capillary DNA sequencer. The heights and areas of
the peaks were checked visually using the GeneMapper software version 3.7
(Applied Biosystems, Foster City, CA, USA) and the data were exported to
an Excel file for further calculations.
3. Results
In one of the patients the earlier reported [4] missense mu-
tation (346C / A or Ala82Glu) in exon 3 of the Parkin gene
was detected. When we investigated the effect of the amino
acid change on protein function by the PolyPhen method
(http://genetics.bwh.harvard.edu/pph/) it was found not to be
damaging. Two novel changes were observed in the coding
parts of the gene; a silent mutation in exon 7 (938C>T or
His288His) found in two patients and a variation in intron
10 (IVS10 þ73G>T) found in two patients. Six earlier
reported single nucleotide polymorphisms (SNPs), in coding
and non-coding parts of the Parkin gene, were also detected
(see Table 1). No deletions or duplications of exons in any
of the investigated genes were found.
4. Discussion
We found one heterozygous carrier of the missense mutation
Ala82Glu among the PD patients. This variation has been
found in earlier studies among both patients and controls [4],
which do not support an involvement of this variation in PD.
Six earlier reported SNPs (see SNP ID, Table 1) with allele
 Letter to the Editor / Parkinsonism and Related Disorders 14 (2008) 520e522 521

Table 1 Foundation, Hållstens forskningsstiftelse, The Swedish Par-

Identified Parkin gene variations in the study sample kinson Foundation, Karolinska Institutet Funds and The
Variation Exon/ SNP ID Number of Minor allele Swedish Brain Power Initiative. The corresponding author
intron patients frequency (%)b had full access to all of the data in the study and takes
IVS2 þ25T>C 2 rs2075923 C: 32 responsibility for the integrity of the data and the accuracy
Ala82Glu 3 1 of the data analysis.
IVS3 20C>T 3 rs4709583 T: 10
His288Hisa 7 2
IVS7 35A>G 7 rs3765474 G: 39
IVS8 þ48C>T 8 rs10945756 T: 22 References
Val380Leu 10 rs1801582 C: 13
IVS10 þ73G>Ta 10 2
[1] Gasser T. Genetics of Parkinson’s disease. Curr Opin Neurol
Asp394Asn 11 rs1801334 A: 4
2005;18(4):363e9.
a
Variations not reported in earlier studies. [2] West AB, Lockhart PJ, O’Farell C, Farrer MJ. Identification of a novel
b
For known polymorphisms. gene linked to Parkin via a bi-directional promoter. J Mol Biol
2003;326(1):11e9.
[3] Daniel SE, Lees AJ. Parkinson’s Disease Society Brain Bank, London:
overview and research. J Neural Transm Suppl 1993;39:165e72.
[4] Kay DM, Moran D, Moses L, Poorkaj P, Zabetian CP, Nutt J, et al.
frequencies in line with previous results were detected [4]. Heterozygous parkin point mutations are as common in control subjects
Further, we identified two novel variations; His288His in as in Parkinson’s patients. Ann Neurol 2007;61(1):47e54.
exon 7 and IVS10 þ73G>T, which probably do not influence [5] Kann M, Jacobs H, Mohrmann K, Schumacher K, Hedrich K, Garrels J,
et al. Role of parkin mutations in 111 community-based patients with
the function of the protein.
early-onset parkinsonism. Ann Neurol 2002;51(5):621e5.
One explanation to the low frequency of mutations in our [6] Lucking CB, Durr A, Bonifati V, Vaughan J, De Michele G, Gasser T,
study might be that our population consists mainly of patients et al. Association between early-onset Parkinson’s disease and mutations
with the sporadic form of PD. However, other studies where in the Parkin gene. N Engl J Med 2000;342(21):1560e7.
the patients were selected in the same way as in the present [7] Carmine Belin A, Westerlund M, Sydow O, Lundstromer K, Hakansson A,
Nissbrandt H, et al. Leucine-rich repeat kinase 2 (LRRK2) mutations in
study [5] reported a higher frequency. A more probable expla-
a Swedish Parkinson cohort and a healthy nonagenarian. Mov Disord
nation to the low mutation rate is the relatively high age of on- 2006;21(10):1731e4.
set of PD in our population, with many individuals reporting
an age of onset between 40 and 50 years. It has been shown Anna Håkansson*
in other studies, for example by [6], that the frequency of Department of Pharmacology,
Parkin mutations in patients with sporadic PD decreased Sahlgrenska Academy at Göteborg University,
significantly with increasing age at onset. P.O. Box 431, S 405 30 Göteborg,
During the MLPA analysis no aberration in the copy num- Sweden
bers of exons in the genes a-synuclein, UCHL-1, DJ-1 and *Corresponding author. Tel.: þ46 31 773 3425;
PINK1 were detected. The presence of the three known fax: þ46 31 82 17 95.
LRRK-mutations R1441G/C/H, G2019S and I2020T have E-mail address: anna.hakansson@pharm.gu.se
been investigated and none of the totally 73 PD patients in-
cluded in the present study carry any of these mutations. How- Andrea Carmine Belin
ever, the G2019S-mutation has been detected among three Department of Neuroscience,
other PD patients from Stockholm and one control individual Karolinska Institutet,
from Göteborg [7]. Stockholm,
In summary our findings do not support a major role for the Sweden
Parkin gene or any of the other investigated genes as the cause Camilla Stiller
of PD among our Swedish, mainly early age of onset, PD Swegene Genomics Core Facility,
patients. Sahlgrenska Academy at Göteborg University,
Göteborg, Sweden
Acknowledgements
Olof Sydow
Department of Clinical Neuroscience,
The skilful technical assistance of Anna Anvret, Marie
Karolinska Institutet,
Carlsson, Brita Eriksson, Lena Espefält, Ann Larsson, Ulla
Stockholm, Sweden
Lennartsson, Karin Lundströmer, Gunilla Nordlund, Håkan
Sandbjörk and Ann-Christin Thelander is gratefully Bo Johnels
acknowledged. We would also like to thank The SWEGENE Department of Clinical Neuroscience and
Göteborg Genomics Core Facility platform which was Rehabilitation, Sahlgrenska Academy at
funded by a grant from the Knut and Alice Wallenberg Göteborg University,
Foundation. The study was supported by The Swedish Göteborg,
Research Council, Åhlén’s Foundation, The Swedish Brain Sweden
522 Letter to the Editor / Parkinsonism and Related Disorders 14 (2008) 520e522

Lars Olson Hans Nissbrandt

Department of Neuroscience, Department of Pharmacology,
Karolinska Institutet, Sahlgrenska Academy at Göteborg University,
Stockholm, Sweden P.O. Box 431, S 405 30 Göteborg,
Sweden
Björn Holmberg
Department of Clinical Neuroscience and Rehabilitation,
6 July 2007
Sahlgrenska Academy at Göteborg University,
Göteborg, Sweden