A comprehensive analysis of deletions, multiplications, and copy number variations in PARK2

Abstract

Objectives: To perform a comprehensive population genetic study of PARK2. PARK2 mutations are associated with juvenile parkinsonism, Alzheimer disease, cancer, leprosy, and diabetes mellitus, yet ironically, there has been no comprehensive study of PARK2 in control subjects; and to resolve controversial association of PARK2 heterozygous mutations with Parkinson disease (PD) in a well-powered study.

Methods: We studied 1,686 control subjects (mean age 66.1 ± 13.1 years) and 2,091 patients with PD (mean onset age 58.3 ± 12.1 years). We tested for PARK2 deletions/multiplications/copy number variations (CNV) using semiquantitative PCR and multiplex ligation-dependent probe amplification, and validated the mutations by real-time quantitative PCR. Subjects were tested for point mutations previously. Association with PD was tested as PARK2 main effect, and in combination with known PD risk factors: SNCA, MAPT, APOE, smoking, and coffee intake.

Results: A total of 0.95% of control subjects and 0.86% of patients carried a heterozygous CNV mutation. CNV mutations found in 16 control subjects were all in exons 1-4, sparing exons that encode functionally critical protein domains. Thirteen patients had 2 CNV mutations, 5 had 1 CNV and 1 point mutation, and 18 had 1 CNV mutation. Mutations found in patients spanned exons 2-9. In whites, having 1 CNV was not associated with increased risk (odds ratio 1.05, p = 0.89) or earlier onset of PD (64.7 ± 8.6 heterozygous vs 58.5 ± 11.8 normal).

Conclusions: This comprehensive population genetic study in control subjects fills the void for a PARK2 reference dataset. There is no compelling evidence for association of heterozygous PARK2 mutations, by themselves or in combination with known risk factors, with PD.

Figure 1 Location of PARK2 copy number variations Deletions (hatched arrows) and multiplications (open arrows) detected among 2,091 patients (top) and 1,686 control subjects (bottom). The PARK2 coding region is shown in gray; the 2 ring domains are shown in black. Introns not drawn to scale. E = exon.

Figure 2 Age at onset of Parkinson disease as a function of number of PARK2 mutations Patients with 2 (long dashed line), 1 (short dashed line), and 0 (solid line) PARK2 mutations. Rare nonsynonymous sequence variants were considered to be a mutation only when found in an individual carrying a copy number variation (CNV). Age at onset in heterozygous PARK2 patients is not earlier than in patients lacking a PARK2 CNV mutation, as opposed to patients with compound mutations who have substantially earlier onset. The graph was generated using Kaplan-Meier survival analysis and the differences were tested using log-rank statistics. Overall, p = 1 × 10⁻⁴⁰, which is driven by compound mutation carriers in recessive young-onset disease.

Figure 3 Moving average plots of PARK2 mutation frequency as a function of age Moving average frequency of PARK2 mutations was estimated as a function of age at onset in patients (red triangles) vs age at blood draw in controls (blue circles) with 95% central posterior probabilities (red and blue lines). Plots show that PARK2 mutation frequency is very high in young-onset Parkinson disease (PD), declines with increasing age at onset, and by age 45 and thereafter, the mutation frequency in PD and control subjects are completely superimposed. Gray bars at the bottom of the graph show the statistical significance of the difference between patients and control subjects, calculated in the moving window (light gray ≥95% probability, dark gray ≥99% probability). Due to few numbers at the extremes, subjects with ages ≤20 or ≥90 years were merged.