The parkin gene is not involved in late-onset Parkinson’s disease

Subjects and methods.

We studied 118 patients with LOPD. All patients had a clinical diagnosis of PD according to the United Kingdom PD Society Brain Bank criteria,4 and an age at onset of 45 years or older. This cutoff was chosen to extend the analysis of the parkin gene to patients older than 45 years, as a previous study had investigated patients younger than 45 years.2 Each subject gave a fully informed consent for the study. The investigated population consisted of two groups: patients with familial LOPD (with a probable autosomal recessive inheritance) and patients with sporadic LOPD.

Twenty-three patients with familial LOPD were screened from 18 families (figure), selected for the study according to the following criteria: reported parkinsonism in two or more siblings; a mode of inheritance compatible with autosomal recessive transmission (affected siblings without affected parents); and an age at onset of 45 years or older in all the affected siblings. All families originated from regions of Southern Italy: 13 families from Calabria were consecutively recruited from our Center at the University of Catanzaro since 1997; 5 other families were recruited in other regions (4 from Sicily and 1 from Puglia). In four families the parents of the affected siblings were consanguineous. Forty-one affected family members were identified, and 23 patients were available for clinical and molecular studies; for the remaining 18 patients (8 were deceased) detailed medical records accounted for their parkinsonism.

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Figure. Simplified pedigrees of 18 families with probable autosomal recessive late-onset PD. Black squares (men) and circles (women) represent affected individuals with age at onset (in years) indicated under the patient’s symbol. Slashed symbols indicate deceased individuals. Asterisks indicate individuals available for clinical and DNA analysis. Numbers of unaffected children are given in diamonds. Double lines indicate consanguineous marriages (families SI-005, CA-009, CA-010, and CA-016).

Ninety-five patients had sporadic LOPD without a family history of PD. They had the same ethnic origin (Calabria, Southern Italy), and were consecutively selected among the outpatients attending the Institute of Neurology of the University of Catanzaro in 1999. Moreover, 100 healthy subjects (52 men and 48 women; mean age ± SD, 68.8 ± 9.1 years) with the same ethnic background served as an age-matched control group.

Genomic DNA was extracted from all subjects from peripheral blood leukocytes according to standard procedures. In all patients with LOPD, the 12 coding exons of the parkin gene were amplified by PCR using the same oligonucleotide primer pairs previously described.5 The PCR products were visualized on ethidium bromide–stained 2% agarose gels and the presence or absence of the target exons was detected. Moreover, in all patients, single-strand conformational polymorphism (SSCP) analysis to predict point mutations was performed on all 12 exons of parkin, as well as flanking intron sequences, using published primers.5 In all 23 patients with autosomal recessive LOPD and in 30 patients with sporadic LOPD, the same primers were used for the sequencing of the PCR products of the 12 exons on both strands using the Big Dye Terminator Cycle Sequencing Ready Reaction DNA Sequencing Kit (Applied Biosystems, Foster City, CA), according to the manufacturer’s recommendations, on an ABI 377 automated sequencer. Nucleotide positions were determined according to the cDNA sequence published in the DNA Data Bank of Japan (DDBJ accession no. AB009973). Additionally, in all patients and control subjects, four previously reported exonic restriction fragment length polymorphisms of the parkin gene were investigated: Ser167Asn, Arg366Trp, Val380Leu, and Asp394Asn.6,7⇓ Exons 4, 10, and 11 of the parkin gene were amplified by PCR using oligonucleotide primer pairs previously described.6,7⇓ The Ser167Asn polymorphism in exon 4 was analyzed by digestion with AlwN I (New England Biolabs, Beverly, MA). The Arg366Trp and Val380Leu polymorphisms in exon 10 were analyzed by digestion with Nci I and Bsp1286 I (New England Biolabs, Beverly, MA), respectively. The Asp394Asn polymorphism in exon 11 was analyzed by digestion with Taq I (Roche, Mannheim, Germany).

Statistical analysis concerning clinical data were carried out with unpaired t-test and with the Mann-Whitney U test, whereas the polymorphisms of the parkin gene were compared between patients and controls with the χ² test with Yates correction and Fisher’s exact probability test under the assumption of Hardy-Weinberg equilibrium.

Results.

Demographic and clinical characteristics of patients with LOPD are shown in table 1. No significant differences were observed between the groups of patients with familial LOPD and patients with sporadic LOPD. All patients presented a good clinical response to levodopa (except three patients with familial LOPD and six patients with sporadic LOPD who were never treated with the drug).

No homozygous exon deletions were found by analysis of PCR products in all patients with LOPD, either familial or sporadic. SSCP and sequence analysis of all coding exons did not reveal any point mutations in the investigated patients. Polymorphisms were found in exon 4 (Ser167Asn), exon 10 (Val380Leu), and exon 11 (Asp394Asn) in some patients with sporadic or familial LOPD. Nevertheless, the allele and genotype frequencies of each tested polymorphism did not significantly differ between patients and controls (table 2).

Discussion.

Our findings indicate that the parkin gene is not involved in the pathogenesis of classic LOPD, either familial recessive or sporadic. Indeed, we did not find any parkin mutations in 18 families with autosomal recessive LOPD or in 95 patients with sporadic LOPD. Moreover, we did not find any associations between four different polymorphisms of the parkin gene and the development of classic LOPD.

A recent study showed that parkin mutations are a major cause of EOPD, a form of parkinsonism with an onset of symptoms before the age of 45 years.2 EOPD, however, accounts for only a minority (about 1/10) of cases with PD8 and, even though mutations in the parkin gene have been also reported in some affected siblings belonging to families with autosomal recessive EOPD who had an age at onset as late as 58 years,7 the contribution of parkin pathology to the most frequent LOPD has not been established before the current study. This study extended the analysis of the parkin gene to sporadic patients and to families in which all the affected members had an age at onset older than 45 years. Our evidence suggests that mutations in the parkin gene are unlikely in LOPD; moreover, our data support the hypothesis that EOPD due to parkin pathology and LOPD are different clinical entities, in agreement with the absence of Lewy bodies in the former condition.3 We are aware that our methods to detect parkin mutations may underestimate the rate of heterozygous exon rearrangements, because we did not use a semiquantitative PCR assay.2 However, as it has been reported that only a minority of patients with EOPD had single or compound heterozygous exon rearrangements, we maintain that, even with the aforementioned bias, our results are valid. Indeed, most patients with EOPD presented other types of mutations, such as homozygous exon rearrangements and homozygous or heterozygous point mutations,2 that could be ascertained with the techniques used in the current study.

Some peculiar polymorphic states of the parkin gene have been recently associated with different risks for the development of sporadic LOPD in Japan, as heterozygosity at codon 167 (Ser/Asn) was associated with an increased risk,9 whereas another polymorphism located in exon 10 (R/W366; Arg366Trp in this study) was associated with a decreased risk.6 Our findings, however, do not confirm those observations,6,9⇓ because we did not find any difference between patients with LOPD and normal controls for the allele and genotype frequencies of the four investigated polymorphisms. Moreover, it should be noted that the allele frequencies in our populations differed from those reported in Japan,6,9⇓ but were similar to the frequencies reported in other European populations,7,10⇓ suggesting that allele frequencies of parkin polymorphisms can be ethnically dependent.