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. Author manuscript; available in PMC: 2016 Feb 25.
Published in final edited form as: J Med Genet. 2009 Apr 6;46(6):375–381. doi: 10.1136/jmg.2008.063917

Parkin and PINK1 mutations in early-onset Parkinson’s disease: comprehensive screening in publicly available cases and control

J Brooks 1, J Ding 1, J Simon-Sanchez 1, C Paisan-Ruiz 2, A B Singleton 1, S W Scholz 1,2
PMCID: PMC4767009  NIHMSID: NIHMS739165  PMID: 19351622

Abstract

Background

Mutations in parkin and PTEN-induced protein kinase (PINK1) represent the two most common causes of autosomal recessive parkinsonism. The possibility that heterozygous mutations in these genes also predispose to disease or lower the age of disease onset has been suggested, but currently there is insufficient data to verify this hypothesis conclusively.

Objective

To study the frequency and spectrum of parkin and PINK1 gene mutations and to investigate the role of heterozygous mutations as a risk factor for early-onset Parkinson’s disease (PD).

Methods

All exons and exon-intron boundaries of PINK1 and parkin were sequenced in 250 patients with early-onset PD and 276 normal controls. Gene dosage measurements were also performed, using high-density single-nucleotide polymorphism arrays.

Results

In total 41 variants were found, of which 8 have not been previously described (parkin: p.A38VfsX6, P.C166Y, P.Q171X, p.D243N, p.M458L; PINK1: p.P52L, P.T420T, P.A427E). 1.60% of patients were homozygous or compound heterozygous for pathogenic mutations. Heterozygosity for pathogenic parkin or PINK1 mutations was over-represented in patients compared with healthy controls (4.00% vs. 1.81%) but the difference was not significant (p = 0.13). The mean age at disease onset was significantly lower in patients with homozygous or compound heterozygous mutations than in patients with heterozygous mutations (mean difference 11 years, 95% Cl 1.4 to 20.6, p = 0.03). There was no significant difference in the mean age at disease onset in heterozygous patients compared with patients without a mutation in parkin or PINK1 (mean difference 2 years, 95% Cl −3.7 to 7.0, p = 0.54).

Conclusions

Our data support a trend towards a higher frequency of heterozygosity for pathogenic parkin or PINK1 mutations in patients compared with normal controls, but this effect was small and did not reach significance in our cohort of 250 cases and 276 controls.

Parkinson’s disease (PD) is a common neurodegenerative disorder characterised by bradykinesia, rigor, tremor and postural instability. Several lines of evidence suggest that genetic susceptibility plays an important role in the pathogenesis of PD.1 Mutations in parkin and PTEN-induced putative kinase 4 (PINK1) have been found to cause early-onset PD (disease onset before the age of 50 years) with autosomal recessive inheritance.2,3 Parkin, encoding the E3 ubiquitin ligase parkin, is one of the largest genes in the human genome, spanning approximately 1.4 Mb. It is located on chromosome 6q25.2-q27 in a highly unstable genomic region.4 Point mutations and exon rearrangements in parkin are the most common genetic causes of early-onset PD; approximately 18% of sporadic and 50% of all familial early-onset cases have been attributed to parkin mutations.5 PINK1, located on chromosome 1p36, has been implicated in PD based on linkage studies in families with autosomal recessive early-onset parkinsonism.2 In contrast to parkin, PINK1 is a much smaller gene, containing only eight exons that span about 18 kb. Homozygous or compound heterozygous loss of function mutations in PINK1 have been found to lead to mitochondrial dysfunction.6,7

There is growing evidence that heterozygosity for parkin or PINK1 mutations might act as a susceptibility factor for PD.8–10 There is an increased frequency of heterozygous mutations in patients with PD compared with healthy controls, and cross-sectional imaging studies have shown subclinical dopaminergic dysfunction in heterozygous parkin or PINK1 mutation carriers.9,11,12 In addition, an inverse relationship has been suggested between the number of pathogenic mutations and the age at disease onset.8,13 However, the literature is not consistent, as some studies report a similar frequency of heterozygous mutations in cases and in controls.14–16 Additional screening studies are therefore required to investigate the relevance of heterozygous pathogenic mutations to the pathogenesis of PD.

We undertook a comprehensive screening study of a large, publicly available cohort of patients with early-onset PD (defined as age at disease onset 50 years) and normal controls, to determine the frequencies and spectrum of mutations in parkin and PINK1 and to investigate the role of heterozygous mutations as a risk factor for PD. We identified several novel mutations of which some are likely to be pathogenic, and found a trend towards a higher frequency of heterozygosity for pathogenic parkin or PINK1 mutations in patients compared with normal controls.

METHODS

All participants gave written informed consent to participate in genetics research and the research protocol was approved by the institutional review board.

Subjects

We screened 250 early-onset patients with PD (mean age at disease onset 41 years, range 7–50) and 276 neurologically normal controls (mean age at sampling 39 years, range 15–53) for mutations in parkin and PINK1. These samples, precompiled in six plates (NDPT14, NDPT15, NDPT16, NDPT19, NDPT20 and NDPT21), are publicly available at the NINDS Neurogenetics Repository at the Coriell Institute for Medical Research, New Jersey, USA (http://ccr.coriell.org/ninds). Clinical characteristics of cases and controls are summarised in table 1.

Table 1.

Clinical description of cases and controls

Characteristics Patients Controls
Subjects, n 250 276
Female/Male   97/153 199/77
Mean age at sampling, years   54   39
Mean age at onset (range), years   41 (7 to 50) MA
Ethnicity
 Caucasian: Hispanic     7     3
 Caucasian: not Hispanic 156 273
 Caucasian: not specified   87     0
Positive family history*   95   44
No information on family history     3     0
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NA, not applicable.

*

Positive family history for one or more of: parkinsonism, tremor, dementia, restless legs syndrome, dystonia.

Sequencing

All exons and exon-intron boundaries of parkin (reference sequence: NM_004562.1) and PINK1 (reference sequence: NM_0032409.2) were amplified by PCR, in a reaction mix of 15 ng of genomic DNA, 10 nmol/μl forward primer, 10 nmol/μl reverse primer and 12 μl of a master mix (FastStart PCR Master; Roche, Indiana, USA). For exon 1 in parkin, 1 μl of 5% dimethylsulphoxide (American Bioanalytical, Massachusetts, USA) was added to the PCR reaction mix and PCR amplification for exon 1 in PINK1 was achieved by adding 12.5 nmol/μl 7-deaza-GTP (New England Biolabs, Massachusetts, USA) to the mix (primer sequences and fragment lengths are shown in supplementary table 1 online). For the PCR reaction, we used a 60 touch down 50 thermo-cycling program. PCR cycling conditions were as follows: initial denaturation step of 4 minutes at 94°C; 8 cycles of 30 seconds at 94°C, 30 seconds at 60°C and 30 seconds at 72°C; 20 cycles of 30 seconds at 94°C, 30 seconds at 60°C, decreasing by 0.5°C after each cycle, 30 seconds at 72°C; 16 cycles of 30 seconds at 94°C, 20 seconds at 50°C and 30 seconds at 72°C; and a final extension step of 5 min at 72°C.

After PCR product purification (AMPure; Agencourt Bioscience Corporation, Massachusetts, USA), we performed direct dye terminator sequencing (BigDye v3.1; Applied Biosystems, California, USA). The resulting reactions were cleaned with CleanSEQ (Agencourt Bioscience Corporation, Massachusetts, USA), processed on an automated analyser (3730 ×l DNA Analyzer; Applied Biosystems) and analysed with Sequencher V.4.1.4 (Gene Codes Corp., Michigan, USA).

For sample ND00548 from plate NDPT14, chromatography analysis showed a complex mutation in exon 2 of parkin. We therefore performed allele-specific sequencing in this sample. Exon 2 was amplified by PCR and then cloned (pCR8/GW/TOPO TA Cloning Kit; Invitrogen, Carlsbad, California, USA), in accordance with the manufacturer’s instructions. Transformed colonies were cultured overnight in LB medium containing 100 μg/ml spectinomycin and followed by isolation of plasmid DNA using PureLink Quick Plasmid Miniprep Kit (Invitrogen). DNA from 12 colonies was sequenced bidirectionally using direct sequencing as described above.

Gene dosage measurements

In all samples we performed genome-wide single-nucleotide polymorphism (SNP) genotyping using SNP chips (Infinium HumanHap 550K V.3; Illumina Inc., California, USA) in accordance with the manufacturer’s protocol. Raw genotypes were determined using the genotyping module of BeadStudio V.3.1.12 (Illumina Inc.). Gene dosage, inferred from signal intensity data and genotype calls at the parkin and PINK1 loci, was visualised using Genome Viewer V.3.1.4 within BeadStudio as previously described.17 A heterozygous duplication was called if the log R ratio (a metric for gene dosage) was increased by 50%, whereas a 50% decrease in the log R ratio in combination with absence of heterozygous SNP calls was considered a heterozygous deletion.

In samples with an exon rearrangement involving the coding sequence of parkin, we performed quantitative PCR (qPCR) assays (TaqMan; Applied Biosystems) to confirm each rearrangement. β-globin was used as an endogenous reference. Each reaction contained two fluorescent-label TaqMan MGB probes (Applied Biosystems), one VIC-labelled probe that specifically bound to the respective parkin exon sequence and one 6-FAM-labelled probe that was specific for the β-globin reference (primer and probe sequences are shown in supplementary tables 2 and 3 online). The qPCR reaction mix consisted of 25 ng genomic DNA, 10 μl of 2×TaqMan Universal PCR Master Mix (Applied Biosystems), 72 nmol/μl primers, 10 nmol/μl of the β-globin probe and 10 nmol/μl test probe. PCR cycling conditions were: 95°C for 10 minutes, 95°C for 15 seconds and 60°C for 1 minute (40 cycles). Each sample was replicated six times and for each exon, two control samples were measured. The dosage of each exon relative to β-globin and normalised to control DNA was determined using the 2−ΔΔCT method.

Bioinformatic analyses

For multiple sequence alignments, human PINK1 and parkin reference sequences were aligned with paralogues and orthologues from the NCBI conserved domain database using ClustalW2. First, the serine/threonine protein kinase catalytic domain (c109925) was identified in the PINK1 sequence by querying NCBI conserved domain database and then sequence-to-structure alignments were applied to the kinase domain of PINK1 to map the kinase functional subdomains to the PINK1 kinase region. Similar approaches were applied on the RING-finger domains of parkin.

Statistical analyses

For sequence analysis, we defined any alteration from the reference sequence as a variant or a mutation. Changes in exon copy number were referred to as exon rearrangements. Polymorphisms were defined as variants that occurred at a minor allele frequency of at least 1% in the control population. To ensure accuracy of the statistical analyses, we only called a mutation pathogenic if one of the following criteria applied: stop mutation, frameshift mutation, exon rearrangement or a missense mutation that has been reported to be pathogenic (details are presented in table 2).

Table 2.

Subjects with pathogenic mutations included in statistical analysis

Sample ID Mutation(s)* Age at onset, years Family history
Pathogenic parkin mutations in cases
 ND00136 Heterozygous for p.Q171X Heterozygous for p.R275W 38 Present
 ND00153 Heterozygous deletion of exons 5–6 29 Present
 ND00187† Heterozygous duplication of exons 5–9 38 Present
 ND00429† Heterozygous duplication of exons 5–9 41 Present
 ND00548 Heterozygous for p.A38VfsX6 Heterozygous for p.Q34RfsX5 36 Absent
 ND01119 Heterozygous deletion exon 2 50 Absent
 ND01122 Heterozygous for p.R275W 47 Absent
 ND02639 Homozygous for p.L112LfsX15 30 Absent
 ND02798 Heterozygous duplication of exon 6 32 Present
 ND04581 Heterozygous deletion exon 2 Heterozygous for p.Q34RfsX5 22 Present
 ND05921 Heterozygous deletion exon 4 49 Present
 ND06330 Heterozygous duplication exons 2–3 50 Absent
 ND06635 Heterozygous for p.N52MfsX29 42 Present
Pathogenic PINK1 mutations in cases
 ND08471 Heterozygous for p.M318L 47 Absent
Pathogenic parkin mutations in controls
 ND03967 Heterozygous deletion exon 3 NA Present
 ND04990 Heterozygous duplication exon 2 NA Absent
 ND08538 Heterozygous deletion exons 2–4 NA Absent
 ND09912 Heterozygous for p.R275W NA Absent
 ND10272 Heterozygous for p.T415N NA Present
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NA, not applicable.

*

Only frameshift mutations, stop mutations, exon rearrangements that span the coding sequence and missense mutations that have been reported to be pathogenic are listed.

†

ND00187 and ND00429 are siblings.

Fisher’s exact tests on allelic association and tests for assessing departures from Hardy–Weinberg equilibrium were performed using PLINK (version 1.04; http://pngu.mgh.harvard.edu/~pur-cell/plink/).18 Statistical significance for differences in age at onset was tested using t tests after Kolmogorov–Smirnov tests showed a normal distribution of the data (SPSS V.12.0.1). For the age at onset analysis, p <0.05 was considered significant.

RESULTS

We found 41 sequence variants in a cohort of 250 patients with early-onset PD and 276 normal controls (fig 1, tables 3,4). No significant departures from Hardy–Weinberg equilibrium were found for any of these variants and none of the identified variants achieved significance for allelic association after Bonferroni correction for multiple testing (adjusted p <0.0012).

Figure 1.

Figure 1

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Missense, nonsense and frameshift mutations observed in this study. Arrows indicate the position of each respective mutation relative to the protein domains. Novel mutations are indicated by red font, whereas previously described mutations are black. UBL, ubiquitin-like domain; RING1, RING finger motif 1; IBR, in-between-RING domain; RING2, ring finger motif 2; MTS, mitochondrion transit sequence.

Table 3.

Variants in parkin in 250 PD cases and 276 controls

Nucleotide Location/rs number Amino acid change Mutation type Genotypes in cases (A1A1/A1A2/A2A2) Genotypes in controls (A1A1/A1A2/A2A2) MAF cases/controls Ref.
Novel mutations that are likely to be pathogenic
c.[214delC+216GR T] Exon 2 p.Ala38ValfsX6 Frameshift 0/1/249 0/0/276 0.002/0.000 –
c.612CR T Exon 4 p.Gln171X Stop 0/1/249 0/0/276 0.002/0.000 –
Novel mutations of unknown significance
C.598GR A Exon 4 p.Cys166Tyr Missense 0/1/249 0/0/276 0.002/0.000 –
C.828GR A Exon 6 p.Asp243Asn Missense 0/0/250 0/1/275 0.000/0.002 –
C.1473AR C Exon 12 p.Met458Leu Missense 0/1/249 0/0/276 0.002/0.000 –
Previously described mutations that are likely to be pathogenic
c.202−203delAG Exon 2 p.Gln34ArgfsX5 Frameshift 0/2/248 0/0/276 0.004/0.000 25
c.256delA Exon 2 p.Asn52MetfsX29 Frameshift 0/1/249 0/0/276 0.002/0.000 25
c.437−477del Exon 3 p.Leu112LeufsX15 Frameshift 1/0/249 0/0/276 0.004/0.000 25
c.924CR T Exon 7/rs34424986 p.Arg275Trp Missense 0/2/248 0/1/275 0.004/0.002 25,27
c.1345CR A Exon 11 p.Thr415Asn Missense 0/0/250 0/1/275 0.000/0.002 25
Previously described mutations with unknown significance
c.802GR A Exon 6 p.Arg234Gln Missense 0/1/249 0/0/276 0.002/0.000 28
c.1305CR T Exon 11 p.Arg402Cys Missense 0/0/250 0/1/275 0.000/0.002 29
c.1390GR A Exon 12 p.Gly430Asp Missense 0/1/249 0/0/276 0.002/0.000 5
c.1444CR T Exon 12 p.Pro437Leu Missense 0/3/247 0/3/273 0.006/0.005 24
Previously described mutations that are likely to be non-pathogenic
c.273−18A Intron 2 – Intronic 0/4/246 0/2/274 0.008/0.004 16
c.34BCR A Exon 3 p.Ala82Glu Missense 0/0/250 0/2/274 0.000/0.004 30,31
c.623CR T Exon 4 p.Leu174Leu Silent 0/1/249 0/2/274 0.002/0.004 15
c.884TR C Exon 7/rs9456711 p.Leu261Leu Silent 0/2/248 0/0/276 0.004/0.000 28,32
Previously described polymorphisms
c.272+25C Intron 2/rs2075923 – Intronic 13/83/154 10/95/171 0.218/0.208 25
c.514−20T Intron 3/rs4709583 – Intronic 1/35/214 1/42/233 0.074/0.080 25
c.601GR A Exon 4/rs1801474 p.Ser167Asn Missense 0/7/243 0/9/267 0.014/0.016 33
c.973−35G Intron 7/rs3765474 – Intronic 52/126/72 52/138/86 0.460/0.438 25
c.1034+48T Intron 8/rs10945756 – Intronic 16/87/147 17/98/161 0.238/0.239 16
c.1239GR C Exon 10/rs1801582 p.Val380Leu Missense 6/78/166 5/89/182 0.180/0.179 25
c.1281GR A Exon 11/rs1801334 p.Asp394Asn Missense 0/16/234 0/14/262 0.032/0.025 25
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MAF, minor allele frequency.

Genotype distribution of the major allele (A2) and minor allele (A1) is described for each variant.

Table 4.

Variants in PINK1 in 250 and 276 controls

Nucleotide Location/rs number Amino acid change Mutation type Genotypes in cases A1A1/A1A2/A2A2) Genotypes in controls (A1A1/A1A2/A2A2) MAF cases/controls Ref.
Novel mutation that is likely to be pathogenic
c.1374CR A Exon 7 p.Ala427Glu Missense 0/2/248 0/0/276 0.004/0.000 –
Novel mutation of unknown significance
c.249CR T Exon 1 p.Pro52Leu Missense 0/1/249 0/0/276 0.002/0.000 –
Novel mutation that is likely to be non-pathogenic
c.1354GR A Exon 7 p.Thr420Thr Silent 0/1/249 0/0/276 0.002/0.000 –
Previously described mutation that is likely to be pathogenic
c.1046AR T Exon 4 p.Met318Leu Missense 0/1/249 0/0/276 0.002/0.000 34,35
Previously described mutations with unknown significance
c.1109GR A Exon 5/rs55831733 p.Ala339Thr Missense 0/2/248 0/0/276 0.004/0.000 34
c.1325GR A Exon 6/rs45478900 p.Gly411Ser Missense 0/0/250 0/1/275 0.000/0.002 36
c.1667GRA Exon 8 p.Asp525Asn Missense 0/1/249 0/0/276 0.002/0.000 37
c.1822AR G Exon 8 p.Ser576Ser Silent 0/1/249 0/0/276 0.002/0.000 34
Previously described mutations that are likely to be non-pathogenic
c.1030GR A Exon 4/rs56200357 p.Arg312Arg Silent 0/1/249 0/0/276 0.002/0.000 2
c.1267TR C Exon 6/rs45499398 p.Asp391Asp Silent 0/1/249 0/0/276 0.002/0.000 34,38
Previously described polymorphisms
c.283CR T Exon 1 p.Leu63Leu Silent 9/81/160 5/78/193 0.198/0.159 34
c.438AR T Exon 1 p.Gln115Leu Missense 0/17/233 0/32/244 0.034/0.058 18
c.482−7A Intron 1/rs2298298 – Intronic 2/48/200 3/63/210 0.104/0.125 34
c.1054−5G Intron 4/rs3131713 – Intronic 2/51/197 2/63/211 0.110/0.121 34
c.111 MR A Exon 5/rs3738136 p.Ala340Thr Missense 1/33/216 0/18/258 0.070/0.033 34
c.1656AR C Exon 8/rs1043424 p.Asn521Thr Missense 15/110/125 21/110/145 0.280/0.275 34
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Genotype distribution of the major allele (A2) and minor allele (A1) is described for each variant.

MAF, minor allele frequency.

Parkin analysis

The following variants were seen in parkin: 4 frameshift mutations, 13 missense mutations, one stop mutation, 5 intronic variants and 2 silent mutations (table 3). Five of these variants have not been previously described (p.A38VfsX6, p.C166Y, p.Q171X, p.D243N, p.M458L; chromatograms of all novel variants are shown in supplementary figs 1 and 2 online). In addition, we identified four patients (1.6%) and two controls (0.7%) with a heterozygous exon deletion, and four cases (1.6%) and one control (0.4%) with a heterozygous exon duplication (table 2, supplementary figures 4–6 online). Nine patients (3.6%) and five controls (1.8%) were heterozygous for pathogenic parkin mutations, three patients (1.2%) were compound heterozygotes as they carried two different pathogenic mutations, and one patient (0.4%) was homozygous for a pathogenic mutation.

PINK1 analysis

We found the following variants for PINK: nine missense mutations, five silent mutations and two intronic variants (table 4). Of these, three mutations have not been previously reported (p.P52L, p.T420T, p.A427G; chromatograms of all novel variants are shown in supplementary fig 3 online). None of the studied subjects had a PINK1 gene duplication or deletion. One patient (0.4%) was heterozygous for a pathogenic mutation in PINK1, whereas none of the controls carried a heterozygous mutation. No subject had pathogenic PINK1 mutations.

Combined analysis

In all, 14 patients (5.6%) and 5 controls (1.8%) had 1 pathogenic mutations in parkin or PINK1, and this difference was significant (p = 0.02). Of the 14 patients with 1 pathogenic mutation, 8 (57.1%) had a family history of parkinsonism, compared with 87 of 250 (34.8%) patients without a pathogenic mutation (missing family history data: 3 patients without a pathogenic mutation (1.2%)). Of 5 controls with a pathogenic mutation, 2 (40.0%) had a positive family history for PD, compared with only 7 of 271 (2.6%) controls without a pathogenic mutation.

Bioinformatic analysis of sequence conservation

To determine the extent of sequence conservation at newly discovered mutated sites, we aligned the human PINK1 and parkin sequences with human domain paralogues and with orthologues from various species. Based on this analysis, at least three variants were probably pathogenic: p.A38VfsX6 and p.Q171X lead to an early termination of the coding sequence, and p.A427E is located in close proximity to the highly conserved serine-threonine kinase activation loop, and it is likely that mutations at this site will interfere with substrate binding to this crucial domain (fig 2).

Figure 2.

Figure 2

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Alignment of PINK1 kinase domain with conserved serine-threonine protein kinases. Sequence alignment between the PINK1 kinase domain and several serine-threonine protein kinase members in the NCBI conserved domain database. The red swirl ribbons and arrowed ribbons stand for the predicted conserved α-helix and β-strand in three dimensional structures, respectively. The mutation p.Ala427Glu lies close to the highly conserved APE triple that is the C-terminal anchor of the activation loop. It is likely that mutations in or close to this crucial domain interfere with substrate recognition.

DISCUSSION

We report the results of a comprehensive screening study for parkin and PINK1 mutations in 250 patients with early-onset PD and 276 normal controls. We identified 41 sequence variants: 8 novel and 33 previously described. Conservation across various species strongly suggests that at least three of the novel mutations are likely to be pathogenic (p.A38VfsX6, p.A171X, p.A427E). In addition to sequence variants, we also found eight cases and three controls with a heterozygous exon rearrangement (table 2).

Overall, patients with pathogenic changes in parkin or PINK1 accounted for 5.6% of all cases in this study. This frequency is lower than previous reports have suggested.5,19 Possible explanations for this discrepancy could be a population-specific variability in allele frequencies, different case selection criteria, different age at onset distributions in the studied cohorts, and different criteria for determining which variant is considered pathogenic.

Increasing evidence indicates a role of heterozygous pathogenic mutations as a susceptibility factor for PD. Positron emission tomography imaging studies have reported a subclinical dopaminergic dysfunction in heterozygous parkin or PINK1 mutation carriers.11,12,20 This observation is supported by transcranial ultrasound studies that reported hyperechogenicity of the substantia nigra, which was shown to depend on mutation status.11,21,22 Furthermore, subclinical sensory abnormalities have been described in patients with heterozygous PINK1 mutations.23 The frequency of heterozygous pathogenic mutations has been reported to be significantly increased in patients with parkinsonism compared with normal controls.9 However, several studies have provided contradictory data.14–16 The lack of cohesion between these studies may have arisen due to the differences in how variants were defined as pathogenic, which determined if they were included in the statistical analysis. For our study, we used conservative criteria, and only included mutations in the analysis that were frameshift or stop mutations, exon rearrangements or mutations that have been reported as probably pathogenic (table 2). Based on these strict criteria, we found an increased frequency of patients with a heterozygous pathogenic mutation compared with controls (4.0% vs. 1.8%), but the difference was not significant (p = 0.13). None of the patients was heterozygous for pathogenic mutations in both genes. Therefore, tests for possible additive effects were not feasible.

Age at onset analysis found a significantly lower mean age at disease onset in patients who were homozygous or compound heterozygous for pathogenic parkin mutations (mean (SD) 32 (7) years) than in patients with a heterozygous pathogenic mutation (43 (8) years) (mean difference 11 years, 95% CI 1.4 to 20.6, p = 0.03). In contrast to previous reports that have shown a significant decrease in age at onset in heterozygous carriers compared with patients without a heterozygous mutation,8,24 our study did not reveal a significant difference (43 (8) years vs. 41 (9) years; mean difference 2 years, 95% CI −3.7 to 7.0, p = 0.54).

A possible limitation of this study is the moderate resolution of genome-wide SNP chips for the identification of exon rearrangements. On average, the Illumina 550K chip contains about 0.35 SNPs per kb at the parkin locus and 0.39 SNPs per kb at the PINK1 locus. Small duplications or deletions could therefore have been missed, and our finding of 11 exon rearrangements in the entire series could be an underestimation.

In conclusion, we present the results of a comprehensive analysis of variation in the parkinsonism genes parkin and PINK1. Our data found that there was a trend towards a higher frequency of heterozygosity for pathogenic parkin or PINK1 mutations in patients compared with controls.

Acknowledgments

We thank the participants of this study and the submitters for depositing samples at the Coriell Institute for Medical Research (http://www.coriell.org/). This research was supported entirely by the Intramural Research Program of the NIH, National Institute on Aging, National Institutes of Health, Department of Health and Human Services, USA (Z-number: Z01 AG000957-05).

Footnotes

Competing interests: None.

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