Alcalay RN, Caccappolo E, Mejia-Santana H, Tang MX, Rosado L, Ross BM, Verbitsky M, Kisselev S, Louis ED, Comella C, Colcher A, Jennings D, Nance MA, Bressman SB, Scott WK, Tanner C, Mickel S, Andrews H, Waters C, Fahn S, Cote L, Frucht S, Ford B, Rezak M, Novak K, Friedman JH, Pfeiffer R, Marsh L, Hiner B, Siderowf A, Ottman R, Marder K, Clark LN. Frequency of Known Mutations in Early-Onset Parkinson DiseaseImplication for Genetic Counseling: The Consortium on Risk for Early Onset Parkinson Disease Study. Arch Neurol. 2010;67(9):1116-1122. doi:10.1001/archneurol.2010.194
To assess the frequency and clinical characteristics of carriers of previously identified mutations in 6 genes associated with early-onset Parkinson disease (PD) and provide empirical data that can be used to inform genetic counseling.
Cross-sectional observational study.
Thirteen movement disorders centers.
Nine hundred fifty-three individuals with early-onset PD defined as age at onset (AAO) younger than 51 years. Participants included 77 and 139 individuals of Hispanic and Jewish ancestry, respectively.
Mutations in SNCA, PRKN, PINK1, DJ1, LRRK2, and GBA were assessed. A validated family history interview and the Unified Parkinson Disease Rating Scale were administered. Demographic and phenotypic characteristics were compared among groups defined by mutation status.
Main Outcome Measure
Mutation carrier frequency stratified by AAO and ethnic background.
One hundred fifty-eight (16.6%) participants had mutations, including 64 (6.7%) PRKN, 35 (3.6%) LRRK2 G2019S, 64 (6.7%) GBA, and 1 (0.2%) DJ1. Mutation carriers were more frequent in those with an AAO of 30 years or younger compared with those with AAO between 31 and 50 years (40.6% vs 14.6%, P < .001), in individuals who reported Jewish ancestry (32.4% vs 13.7%, P < .001), and in those reporting a first-degree family history of PD (23.9% vs 15.1%, P = .01). Hispanic individuals were more likely to be PRKN carriers than non-Hispanic individuals (15.6% vs 5.9%, P = .003). The GBA L444P mutation was associated with a higher mean Unified Parkinson Disease Rating Scale III score after adjustment for covariates.
Individuals of Jewish or Hispanic ancestry with early-onset PD, those with AAO of 30 years or younger, and those with a history of PD in a first-degree relative may benefit from genetic counseling.
Early-onset Parkinson disease (PD) may be defined as disease onset before age 40 or 50 years.1- 3 Mutations in several genes have been associated with early-onset PD, including α-synuclein (SNCA), Parkin (PRKN), phosphatase and tensin homolog–induced putative kinase 1 (PINK1), DJ1, leucine-rich repeat kinase 2 (LRRK2), and glucocerebrosidase (GBA).4 Study inclusion characteristics such as age at onset (AAO), family history of PD, and ethnicity influence the prevalence of specific mutations.5 Ideally, the frequency of specific mutations in early-onset PD would be assessed in a population-based sample. The incidence rate of early-onset PD is estimated at 3 per 100 000 people per year,6 making a population-based genetic investigation impractical, and there are no population-based studies of genetic contributions to early-onset PD. All but 3 published studies7- 9 of the genetic contributions to early-onset PD have focused on familial PD10- 13 or were studies of sporadic PD focusing only on a single gene: PRKN,14PINK1,15DJ1,16LRRK2,17 and GBA.18 A recent Dutch early-onset PD study evaluated 187 subjects with an AAO of less than 51 years for mutations in SNCA, PRKN, PINK1, DJ1, and LRRK2 and found 4% (n = 7) who carried an identified mutation8; however, GBA was not evaluated in that study. The goal of the present study was to examine carriers of genetic mutations in early-onset PD recruited without regard to family history of PD. Herein, we present the frequency and the clinical characteristics of carriers of known mutations in 953 subjects who were clinically evaluated and evaluated as part of the Consortium of Risk for Early Onset Parkinson Disease (CORE-PD) study, a multicenter study of subjects with PD ascertained solely because their AAO was younger than 51 years rather than being based on family history of PD.
Subjects with AAO younger than 51 years (n = 953) included consecutive patients with early-onset PD who met research criteria for PD (UK brain bank)19 in 13 sites participating in the CORE-PD study.20 A Mini-Mental State Examination21 score higher than 23 was required for study inclusion so that accurate self-report data could be obtained. Institutional review boards at all participating sites approved the protocols and consent procedures. Data collected included demographic information, AAO, Unified Parkinson Disease Rating Scale (UPDRS)22 score in the “on” state, completed by a movement disorders specialist, Mini-Mental State Examination, and a family history of PD using a previously validated interview23; however, family history of PD was not an inclusion criterion. Ethnic group was documented by self-report using the format of the 1990 US census.24 A blood sample—from which DNA was isolated—was sent to the National Institute of Neurological Disorders and Stroke Human Genetics Resource Center DNA and Cell Line Repository (http://ccr.coriell.org). Examiners were unaware of subjects' genetic status. Subjects were subsequently divided into mutation groups based on their genetic status. Cases were classified into motor subtypes based on previously described methodology: tremor dominant, postural instability and gait difficulty, or intermediate.25
Subjects were screened for PRKN mutations by denaturing high-performance liquid chromatography (WAVE Transgenomic) as previously described.26 Amplicons were either directly sequenced (n = 126) or analyzed (n = 827) using a PRKN genotyping array27 in DNA samples with abnormal denaturing high-performance liquid chromatography elution profiles. Cycle sequencing was performed on the purified polymerase chain reaction product as per the manufacturer's instructions (BigDye, Applied Biosystems). Products were processed on an ABI3700 or ABI3730 genetic analyzer. Chromatograms were viewed using Sequencher (Genecodes) and sequence variants were determined. To identify genomic deletions and exon rearrangements in PRKN, semiquantitative multiplex polymerase chain reaction was performed as previously described.28 Further details are described elsewhere.29
For analysis of LRRK2 mutations, DNA samples from most cases (n = 515) were analyzed using a previously described genotyping array (Asper Biotech, Tartu, Estonia).27 For the remaining 438 cases, genotyping was performed using matrix-assisted laser desorption/ionization time of flight mass spectrometry (Sequenom, San Diego, California) as previously described.5 All samples analyzed by either the matrix-assisted laser desorption/ionization time of flight or the genotyping array were examined for the mutations G2019S, R1441C, I2020T, and Y1699C. Samples analyzed with the genotyping array were also tested for the G2385R variant.
Ninety cases were previously described and complete sequencing of the GBA gene was performed.20 The remaining cases (n = 863) were analyzed for L444P and N370S mutations: 515 cases were genotyped using the genotyping array, and the remaining cases (n = 348) were analyzed by direct sequencing using previously described methods.20
A subset of 515 cases was analyzed using a genotyping array for point mutations in SNCA, DJ1, and PINK1. The point mutations assessed included A157T, A88P, and E136K in SNCA ; L166P, M26I, D149A, and A104T in DJ1 ; and W437X and G309D in PINK1.
Participants were divided into 6 groups based on mutation status: (1) noncarriers of mutations of any of the genes, (2) PRKN heterozygotes, (3) PRKN homozygotes and compound heterozygotes combined, (4) LRRK2 G2019S carriers, (5) GBA N370S carriers, and (6) GBA L444P carriers. DJ1 mutation carriers, LRRK2 G2385R carriers, and carriers of mutations in more than 1 gene were too rare to be analyzed. Instead, carriers of these mutations are described clinically. Demographic and disease characteristics of the 6 groups were compared using analysis of variance and χ2 tests as appropriate. To test whether mutation status (eg, PRKN) is associated with severity of motor impairment, we used a linear regression model in which UPDRS-III score was the dependent variable and several mutations were independent predictors (compared with noncarriers), adjusting for disease duration, sex, age, daily levodopa dose, and history of surgical intervention for PD. Mutation carrier frequency was then assessed in strata defined by decade of AAO (<20, 21-30, 31-40, and 41-50 years) and by Jewish ancestry (defined as having at least 1 Jewish grandparent, given the high LRRK2 and GBA mutation frequency previously reported among Jews30).
One hundred fifty-eight subjects (16.6%) carried a genetic mutation: 33 were PRKN heterozygotes and 27 were PRKN homozygotes/compound heterozygotes.29 Thirty-one were LRRK2 G2019S carriers (30 heterozygotes and 1 homozygote). One subject carried the LRRK2 G2385R variant; no other pathogenic LRRK2 mutations were found. Fifty-eight were GBA mutation carriers: 18 L444P heterozygotes, 38 N370S heterozygotes, and 2 N370S homozygotes. One subject (previously described16) had a single A104T DJ1 mutation. No carriers of the tested SNCA or PINK1 mutations were identified.
Demographic and disease characteristics of the carriers are described in Table 1. Carriers of PRKN mutations were more likely to be Hispanic and to have an earlier AAO than noncarriers. Motor phenotype was computed in 707 subjects who completed the UPDRS-II and UPDRS-III. LRRK2 G2019S carriers were more likely to manifest the postural instability and gait difficulty motor phenotype.25GBA L444P carriers had higher scores on the UPDRS-III than noncarriers, indicative of more significant motor impairment. In a linear regression model, L444P mutation status was associated with worse motor performance, as represented in a higher UPDRS-III score (P = .007), after adjustment for age, sex, and disease duration, daily levodopa dose, and history of surgical intervention for PD (analysis included 738 cases on whom data on all covariates were available). There was no significant difference in the presenting symptom of PD among the groups: 44% presented with rest tremor, 13% with stiffness, and 7% with action tremor. Six percent reported symmetric findings at presentation.
Demographic and disease characteristics of 7 individuals who carried more than 1 mutation are described in Table 2. None of these subjects reported a history of PD in a first-degree relative.
The distribution of mutation carriers by AAO is presented in Table 3. Overall, mutation frequency increased with younger AAO (P < .001). Mutation carriers were more frequent among cases with AAO of 30 years or younger than among cases with AAO between 31 and 50 years (40.6% vs 14.6%; P < .001, Fisher exact test). Age at onset was significantly younger among PRKN mutation carriers than among carriers of all other mutations (P < .001).
Hispanic individuals were more likely than non-Hispanic individuals to be PRKN carriers (15.6% vs 5.9%; P = .003, Fisher exact test). One hundred thirty-nine subjects (14.6%) reported Jewish ancestry (126 reported that all 4 grandparents were Jewish, and 2 reported 3 grandparents, 9 reported 2 grandparents, and 2 reported 1 grandparent of Jewish ancestry). Overall, mutations were more common in individuals with Jewish ancestry than in those without (32.4% vs 13.7%; P < .001, Fisher exact test). The association was driven by LRRK2 G2019S carriers and GBA N370S carriers who were more likely to be Jewish than not (Table 4). Of the 15 G2019S carriers who did not report Jewish ancestry, data on grandparents' country of origin were available in 11 cases. Four G2019S carriers reported southern European descent (Italy or Portugal) and 4 carriers reported mixed European descent (Germany, Hungary, Ireland, Scotland, and England). One reported eastern European ancestry (Russia and Latvia) and 2 carriers reported Puerto Rican ancestry.
In the entire cohort, mutations were more common in subjects with a history of PD in a first-degree relative than in those without (23.9% vs 15.1%; P = .01, Fisher exact test) (Table 4). However, this association was not apparent in Jews, of whom 31.8% of those with a family history and 32.2% of those without a family history carried an identified mutation (P > .99, Fisher exact test).
This large, uniformly clinically characterized sample of subjects with early-onset PD provides an estimate of the frequency of mutation carriers at movement disorder centers in tertiary referral settings. Although these data were not derived from a population-based sample, the results may guide clinicians in their assessment of the need for genetic counseling, particularly in specific racial/ethnic groups. We have provided reference tables that stratify mutation frequency by AAO, family history of PD, and Jewish ancestry. PRKN carriers were more likely to report a family history of PD than LRRK2 carriers, despite the fact that LRRK2 is autosomal dominant and PRKN is considered a recessive disorder. This finding could be explained by siblings of PRKN carriers who may develop PD at a younger age than siblings of other mutation carriers (ie, LRRK2 siblings may develop the disease at a later age) and by incomplete penetrance of the G2019S mutation.17 Also, the high frequency of PRKN heterozygotes may support the notion that even a single PRKN mutation is a risk of early-onset PD.29 In addition to demographic differences among mutation carriers, we found that LRRK2 G2019S carriers were more likely to manifest the postural instability and gait difficulty motor phenotype,31 and GBA L444P carrier status was associated with a higher UPDRS-III score (implying worse motor function) compared with noncarrier status. While the odds of developing PD may be greater in L444P carriers than in N370S carriers,30 this is the first report of a difference in severity of motor signs among PD cases with different GBA mutations. Larger studies, including examinations in the “off” state, are required to assess whether this statistical difference is a true clinical phenotype or a consequence of multiple comparisons.
The higher frequency of mutations detected herein (16.6%) compared with a previous Dutch study (4%)8 may be explained by the inclusion of GBA genotyping (which accounts for 40% of the mutations detected herein) and by the higher proportion of subjects of Jewish and Hispanic origin in our study. These populations are known to have a higher risk of GBA and LRRK2 (Jewish and Mediterranean basin) and PRKN (Hispanic) mutations.29 Previously, when a subset (n = 90) of our early-onset PD sample (AAO of ≤50 years) was compared with late-onset PD (n = 185, AAO >50 years), GBA mutations were more prevalent in early-onset PD.5,20
The high proportion of subjects with identifiable genetic mutations in GBA, LRRK2, and PRKN emphasizes the importance of these genetic factors in the etiology of early-onset PD. Despite the growing evidence that early-onset PD frequently has a genetic basis, the role of clinical testing remains controversial.32 Clinical testing may help to explain the etiology of PD in an individual with early-onset PD and may be particularly useful for that purpose in patients of Jewish or Hispanic ancestry, those with onset younger than age 30 years, and those with a history of PD in a first-degree relative. Finally, although the probability that an identified genetic mutation has been transmitted by an affected parent to a child can be calculated, the probability that that child will one day develop PD (penetrance) remains unknown for any of these mutations. Therefore, there is little or no role for genetic testing for PD in family planning, predictive, or prenatal examinations at this time. There has been a single study on attitudes toward genetic testing in PD33 and no studies on the influence of PD mutation carrier status on reproductive choices.
We have previously reported the frequency of LRRK2,31PRKN,29 and a subset of GBA carriers separately. Simultaneous comparison among carriers and GBA, SNCA, and PINK1 genetic data is unique to this study. A limitation of this study is its lack of inclusion of cognitively impaired cases, as we only included patients with Mini-Mental State Examination scores greater than 23. Each gene was not completely sequenced. The PRKN gene was completely sequenced in 126 of 953 cases, and only selected mutations were examined in LRRK2, GBA, PINK1, SNCA, and DJ1. Therefore, we may have underestimated the proportion of mutation carriers in the genes we studied, particularly GBA in people who are not of Jewish ancestry.34 We did not examine the PARK9 gene; however, it is rarely associated with early-onset PD.35
In summary, individuals with early-onset PD of Jewish or Hispanic ancestry, those with AAO of 30 years or younger, and those with a history of PD in a first-degree relative may benefit from genetic counseling. Because the vast majority of individuals did not carry a known mutation, additional studies are needed to identify additional genetic and environmental risk factors in this population.
Correspondence: Karen Marder, MD, MPH, Department of Neurology, Columbia University, 630 W 168th St, PH19, Room 120, New York, NY 10032 (email@example.com).
Accepted for Publication: January 6, 2010.
Author Contributions:Study concept and design: Ottman, Marder, and Clark. Acquisition of data: Mejia-Santana, Rosado, Verbitsky, Kisselev, Comella, Colcher, Jennings, Nance, Bressman, Scott, Mickel, Andrews, Waters, Fahn, Cote, Frucht, Ford, Rezak, Novak, Friedman, Pfeiffer, Marsh, Hiner, Siderowf, Ottman, and Clark. Analysis and interpretation of data: Alcalay, Caccappolo, Tang, Ross, Louis, Nance, Bressman, Tanner, and Ottman. Drafting of the manuscript: Alcalay, Ross, and Marder. Critical revision of the manuscript for important intellectual content: Caccappolo, Mejia-Santana, Tang, Rosado, Verbitsky, Kisselev, Louis, Comella, Colcher, Jennings, Nance, Bressman, Scott, Tanner, Mickel, Andrews, Waters, Fahn, Cote, Frucht, Ford, Rezak, Novak, Friedman, Pfeiffer, Marsh, Hiner, Siderowf, Ottman, and Clark. Statistical analysis: Alcalay, Tang, Louis, Scott, Andrews, and Ottman. Obtained funding: Scott, Fahn, Ottman, Marder, and Clark. Administrative, technical, and material support: Caccappolo, Mejia-Santana, Rosado, Ross, Verbitsky, Comella, Colcher, Jennings, Bressman, Scott, Mickel, Frucht, Novak, Friedman, and Siderowf. Study supervision: Andrews and Hiner.
Financial Disclosure: Dr Waters has received speaking honoraria from Teva, Novartis, and BI; she is on the advisory boards for Novartis and Teva; and she has received research funding from Novartis, Solvay, BI, and Schwartz. Dr Fahn has received consulting fees and advisory board membership honoraria from IMPAX Pharma, Boehringer-Ingelheim, Vernalis, Intec Pharma, Merz Pharma, RJG Foundation, Oxford Biomedica, and Proctor-Goodwin; he directs the Columbia University Parkinson's Disease Research Center and Fellowship Training Program, which receives support from the Parkinson's Disease Foundation; he has received grant support from the Smart Family Foundation and the US Department of Defense's Telemedicine and Advanced Technology Research Center for the World Parkinson Congress 2010; he has received lecture honoraria from Boehringer-Ingelheim, Movement Disorder Society, American Academy of Neurology, Columbia University, Sun Pharmaceutical India, and World Association of Sleep Medicine; and he has received honoraria for serving as co-editor of Current Neurology and Neurosurgery Report and for co-authoring Principles and Practice of Movement Disorders. Dr Pfeiffer has received royalties for serving as an editor for Butterworth Heinemann, CRC Press, and Humana Press; he has received lecture honoraria from GlaxoSmithKline, Boehringer-Ingelheim, Novartis, and Teva; he has received consulting honoraria from Boehringer-Ingelheim, Kyowa, Ipsen, Solvay, Theravance, Genactics, and Schlesinger Associations; he has received research grant support and/or contracts from Kyowa, Novartis, Boehringer-Ingelheim, Eisai, UCB/Schwartz, and Santhera; he has received legal consulting fees from Spriggs & Hollingsworth and Davis Graham & Stubbs; and he has served as an editor for Parkinsonism and Related Disorders.
Funding/Support: This study was funded by grants NS36630, UL1 RR024156 (Dr Marder), NS050487, NS060113 (Dr Clark), and P50 NS039764 (Dr Scott) from the National Institutes of Health, and the Parkinson's Disease Foundation (Drs Marder, Fahn, and Clark). Dr Alcalay was supported by a Parkinson's Disease Foundation H. Houston Merritt Fellowship in Movement Disorders.
Additional Contributions: Paul Greene, MD, and Diana Ruiz, BS, provided assistance.