Biochemical and Biophysical Research Communications
Analysis of eighteen deletion breakpoints in the parkin gene
Introduction
Parkin has been identified as the causative gene of autosomal-recessive juvenile parkinsonism (AR-JP) [1]. It is a gigantic gene occupying a 1.4-Mb genomic DNA sequence and consists of 12 exons with a 1.4-kb coding sequence [2]. The gene encodes a type of ubiquitin ligase (E3), which is associated with ubiquitin-conjugating enzymes (E2s) UbcH7 or UbcH8 [3]. Some proteins such as the O-glycosylated form of α-synuclein [4] and the Pael receptor [5] serve as substrates for parkin and accumulate in parkin-deficient patients.
Parkin mutations frequently cause early onset Parkinson’s disease, especially in the case of a family history and an autosomal-recessive mode of transmission. The frequency of such mutations has been estimated as 40–50% in familial cases and 10–20% in idiopathic cases of early onset Parkinson’s disease [6]. In our initial screening of the parkin mutations in Japanese patients with AR-JP, we found frequent occurrences of exon 3, exon 4, or both exon 3 and exon 4 deletions [7]. To date, 95 different mutations have been reported, and the exonic deletions have been commonly observed worldwide [8]. To precisely analyze the deleted regions, we had previously determined the 1.4-Mb genomic DNA sequence of the parkin gene in collaboration with the Sanger Centre [2]. Thus far, the DNA sequences of deletion breakpoints have been reported for only two cases of exon 4 deletions [9]. In this study, we investigated the deletion breakpoints of parkin mutations in 22 families with AR-JP and examined the possible association between these deletion events and meiotic recombinations.
Section snippets
Materials and methods
AR-JP families. This study included 16 Japanese families, one Korean family, one Taiwanese family, one Israeli family, and three Turkish families, totally comprising 27 individuals with AR-JP. Molecular analysis was performed for the 22 unrelated families, and all the patients showed some exonic deletions of the parkin gene. The findings on five of these families have been reported previously [7]. DNA samples were obtained from the patients following informed consent, and the study was approved
Features of the deletion breakpoints
We designed primer sets of 191 sequence tagged sites (STSs) along with the 1.4-Mb genomic sequence of the parkin gene and examined for the presence or absence of each STS in the DNA samples from 27 patients with AR-JP. PCR analysis revealed that all these patients had homozygous deletion of at least one exon. Therefore, they represented 44 deletions (88 deletion breakpoints). At least half of these deletion breakpoints (44 deletion breakpoints) were located (Fig. 1). We found six patients
Acknowledgments
We thank the patients and their family members for participating in this study and Dr. T Sasaki for his assistance. This study was partly supported by grants from the Research for the Future program by the Japan Society for the Promotion of Science (JSPS); the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT); and the Japanese Ministry of Health, Labor and Welfare.
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