Author:
Dominique J. Verlaan Ph.D.^1,2, Tadeu Fantaneanu B.Sc.1, Inge A. Meijer Ph.D.¹, Daniel L. Rochefort M.Sc.¹, Mélanie Bénard B.A.¹, Rosette Jabbour M.D.³, Guy A. Rouleau M.D., Ph.D.¹

¹Faculte de Medecine, Universite de Montreal, Centre de recherche du CHUM, Hopital Notre-Dame-CHUM, Montreal, Quebec, Canada.
²Department of Human Genetics, McGill University, Montreal, Quebec, Canada.
³ Department of Internal Medicine, American University of Beirut Medical Center, Beirut,
Lebanon.

Correspondence:
Dr. Guy A. Rouleau, Centre de recherche du CHUM, Hôpital Notre-Dame, 1560 rue Sherbrooke Est, Bureau Y-3633, Montréal, Québec H2L 4M1, Canada

Introduction

Parkinson disease (PD; MIM# 168600) is a prevalent age-associated progressive neurodegenerative disorder first described by James Parkinson in 1817. After Alzheimer's disease, it is the second most common neurodegenerative disorder, affecting 2% of the population who are over the age of 65 ¹. It is characterized by a combination of resting tremor, bradykinesia and rigidity and postural instability. Other features may include dementia, dystonic cramps and dysautonomia. Idiopathic PD onset usually occurs in mid to late adulthood. Patients frequently have an excellent initial symptomatic response to levodopa therapy; however, it does not slow PD progression and it may also provoke undesirable side effects, such as dyskinesia ^2,3. Pathologically, there is a loss of dopaminergic neurons in the substantia nigra (SN)⁴. In addition, Lewy bodies, which are intracellular inclusions, are present in the surviving neurons mainly in the SN but also in other areas of the brain⁴.

The aetiology of the disease is still largely unknown, but genetic susceptibility factors in some families are strongly suspected. To date, seven autosomal dominant loci including mutations in four genes have been identified: PARK1/PARK4 on chromosome 4q21 (SNCA gene)⁵, PARK3 on 2p13⁶, PARK5 on 4p14 (UCHL1 gene)⁷, PARK8 on 12q12 (LRRK2 gene)⁸, PARK10 on 1p⁹, PARK11 on 2q¹⁰and PARK13 on 2p12 (HTRA2 gene)¹¹. Three autosomal recessive loci and their respective genes have also been described: PARK2 on chromosome 6q25.2-q17 (PARK2 gene)¹², PARK6 on 1p36 (PINK1 gene)¹³ and PARK7 on 1p36 (DJ1 gene)¹⁴.

We identified a large consanguineous Lebanese family affected with adult onset Parkinson's disease. The family consists of three sisters who became affected with PD in their late twenties and that have the classical triad of features of bradykinesia, rigidity, and tremor (Table 1 and Figure 1). The sisters responded very well to levodopa therapy. The unaffected parents are 1st degree cousins and for this reason, loci that are known to cause recessive PD - PARK2, PARK6 and PARK7 - were first investigated by genotyping.

Table 1: Clinical information

Y: yes, N: no

Materials and Methods

Patients: Informed consent was obtained from all patients and family members. Blood samples were collected from each subject and DNA was extracted from peripheral blood by standard methods. Clinically, the three affected subjects were first investigated for Wilson's disease by testing their ceruloplasmin level, serum copper and 24-hours copper in urine, which were normal. Magnetic Resonance Imaging (MRI) of the brain was performed and was also normal.

Linkage analysis and haplotyping: Linkage was performed using polymorphic markers obtained from the Marshfield genetic map, and the primer sequences were obtained from the Genome Database and the Cooperative Human Linkage Center database. Each primer pair was amplified according to specific Polymerase Chain Reaction (PCR) conditions and was labelled by incorporating the nucleotide S35-dATP in the product. The PCR products were separated on 6% denaturing polyacrylamide gels and detected by exposure to autoradiographic film. The alleles were assigned on the basis of their size, with comparison to a M13mp18 sequence ladder. Marker location was obtained from the UCSC physical map (March 2006 Assembly, NCBI build 36.1). D6S1579, D6S1550, D6S305, D6S1599 and D6S1277 were analyzed for the PARK2 locus; D1S2694, D1S548 and D1S1612 were analyzed for the PARK6 locus; and D1S199, D1S3720, D1S2864, D1S482 and D1S2674 were analyzed for the PARK7 locus.

Gene analysis: Each of the 12 PARK2 exons was amplified by PCR with intronic primers and was sequenced on an ABI3700 automated sequencer using BigDye chemistry, according to the manufacturer's recommended protocol (Applied Biosystems, Foster City, CA).


Results

Haplotype analysis showed that there was no segregation of the disease with a haplotype at the PARK6 and PARK7 loci (data not shown). In contrast, all affected individuals were homozygous for the same haplotype (Figure 1) at the PARK2 locus. In addition, all of the normal individuals were heterozygous for the disease haplotype or did not carry it at all. A haplotype recombination event was found in Individual II:10, suggesting that a causative mutation would not be found in the first six exons of the PARK2 gene.
Mutation analysis of the PARK2 gene revealed a single base pair deletion of a guanine at nucleotide 1081 at the end of exon 9. This deletion was found in a homozygous state in all three affected sisters (Figure 2). This mutation presumably leads to a frameshift that introduces a premature stop codon leading to a novel C-terminus containing 72 new amino acids.

Figure 1: Haplotype analysis of the consanguineous Lebanese family at the PARK2 locus. A black bar represents the disease haplotype. Parentheses represent inferred alleles
Black symbol: affected; White symbol: normal.

Figure 2:
a) Normal PARK2 sequence showing the junction between exon 9 and intron 9
b) Normal sequence of a control
c) Mutated sequence in a homozygous state of an affected individual with a deletion of a guanine at nucleotide 1081 (1081delG). This is a frameshift mutation, which leads to a premature stop codon.

Discussion

We have identified a consanguineous Lebanese family that is affected with early onset Parkinson Disease and which segregates a mutated PARK2. An extensive review of the literature suggests that this novel 1081delG mutation is the first PARK2 mutation described in a Lebanese family. Our results support the evidence that PARK2 mutations are very prevalent in PD and are present in nearly every ethnicity¹⁵. The likelihood of carrying PARK2 mutations in individuals with parkinsonism is inversely associated with age and may be as high as 50% in individuals who are younger than 25 years old¹⁶. Interestingly, PARK2 mutations may not only cause early-onset PD but may also be involved in later-onset PD (greater than 60 years) when individuals only have one mutated copy^{17, 18}.

The PARK2 gene, which is mutant in autosomal recessive juvenile parkinsonism (PDJ) (OMIM: 600116) maps to 6q26, contains 12 exons and spans 1.3Mb of genomic DNA12. PARK2, which is one of the largest genes in the human genome, lies within the hyper-recombinable fragile site FRA6E¹⁹. All types of mutation have been identified in the PARK2 gene: multiplications, small deletions/insertions, large genomic deletions as well as splice, missense and nonsense mutations. Most of the missense mutations occur within consensus domains of the gene and affect amino acids which are usually conserved in the mouse. The PARK2 gene encodes for a 465 amino acid protein called parkin, which includes an ubiquitin homologous domain in its N-terminus and two RING finger domains in its C-terminus. Parkin functions as an E3 ubiquitin ligase²⁰ and may interact with alpha-synuclein (SNCA), a protein which is also involved in PD²¹. The protein resulting from the 1081delG mutation would presumably be lacking the last RING domain and contain a disrupted IBR domain.

Lastly, it will be interesting to ascertain if this mutation can also be found in other early-onset PD Lebanese families and in patients with an older age of onset, as well as PD patients from other ethnic origins. In addition, further characterization of this mutation could potentially lead to the development of PD diagnostic and prenatal diagnostic tools for the Lebanese population.

We would like to especially thank the family for participating in this study. DJV is supported by a Quebec Health Research Fund Training and Support for Research scholarship. GAR is supported by the Canadian Institute of Health Research and the Quebec Health Research Fund.