LRRK2 G2019S mutation and Parkinson s disease: A clinical, neuropsychological and neuropsychiatric study in a large Italian sample

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1 Parkinsonism and Related Disorders 12 (2006) LRRK2 G2019S mutation and Parkinson s disease: A clinical, neuropsychological and neuropsychiatric study in a large Italian sample Stefano Goldwurm a,, Michela Zini a, Alessio Di Fonzo b,c, Danilo De Gaspari a, Chiara Siri a, Erik J. Simons b, Marina van Doeselaar b, Silvana Tesei a, Angelo Antonini a, Margherita Canesi a, Anna Zecchinelli a, Claudio Mariani a, Nicoletta Meucci a, Giorgio Sacilotto a, Roberto Cilia a, Ioannis U. Isaias a, A. Bonetti a, Francesca Sironi a,d, Sara Ricca a,d, Ben A. Oostra b, Vincenzo Bonifati b,e, Gianni Pezzoli a a Parkinson Institute, Istituti Clinici di Perfezionamento, Milan, Italy b Department of Clinical Genetics, ErasmusMC Rotterdam, The Netherlands c Centro Dino Ferrari, Department of Neurological Sciences, University of Milan, and Foundation Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milan, Italy d Molecular Genetics Laboratory, Foundation Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milan, Italy e Department of Neurological Sciences, La Sapienza University, Rome, Italy Received 27 January 2006; received in revised form 4 April 2006; accepted 4 April 2006 Abstract We analysed the Leucine-Rich Repeat Kinase 2 (LRRK2) gene for the G2019S mutation in 1245 consecutive, unrelated patients with primary degenerative parkinsonism, and collected information on medical history, motor, cognitive and neuropsychiatric functions to characterize the clinical phenotype associated to the G2019S mutation. The mutation was detected in heterozygous state in 19 probands (1.7%), and in five additional affected relatives. Clinical features in carriers were those of typical, idiopathic Parkinson s disease. However, behavioural abnormalities were frequent (87%), suggesting a more widespread limbic involvement in G2019S carriers. r 2006 Elsevier Ltd. All rights reserved. Keywords: Parkinson; Park8; LRRK2; G2019S mutation; Neuropsychological study 1. Introduction The aetiology of Parkinson s disease (PD) has not been fully elucidated. PD is generally considered to be the result of the interaction between genetic and environmental factors [1]. In recent years many discoveries have been made on the genetic aspects of this Corresponding author. Tel.: ; fax: address: goldwurm@parkinson.it (S. Goldwurm). disease. Several genes involved in familial PD aetiology have been identified (SNCA, parkin, DJ-1, PINK1, LRRK2) and their study has given an important contribution to the understanding of molecular mechanisms underlying the disease [2,3]. The most recent gene identified is the Leucine-rich Repeat Kinase 2 (LRRK2) at the PARK8 locus (OMIM #607060). PARK8 was mapped on the 12q12 band for the first time in a Japanese family with autosomal dominant parkinsonism [4]. The LRRK2 gene was subsequently identified in Caucasian PD families in linkage with PARK8 locus [5,6]. The function of the protein encoded by LRRK /$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi: /j.parkreldis

2 S. Goldwurm et al. / Parkinsonism and Related Disorders 12 (2006) (also termed dardarin), and the mechanism whereby LRRK2 mutations causes PD are unknown. The LRRK2 protein belongs to the ROCO group within the Ras/GTPase super-family, and contains several conserved domains: a Roc (Ras in complex proteins) and a COR (C-terminal of Roc) domain, together with a leucine-rich repeat, a WD40 domain, and a protein kinase catalytic domain [7]. Several mutations co-segregate with PD in families, but the most common is a G4A transition that causes the G2019S missense mutation [8 12]. This mutation has been found in familial as well as sporadic PD, albeit with different frequencies in the different populations [13 19]. A detailed clinical, as well as pathological characterization of PD patients carrying the G2019S and other LRRK2 mutations is now warranted in order to investigate of whether LRRK2-related disease represents the classical PD or a different entity. We analysed the LRRK2 gene for the most frequent mutation, G2019S, in a series of 1245 patients affected by PD or other forms of degenerative Parkinsonism attending a single Centre. The genetic results concerning the first 629 patients, in whom additional recurrent LRRK2 mutations were sought, have already reported previously [20]. Here we report the results of the genetic screening of the whole sample of 1245 patients, as well as the results of a very detailed assessment of PD history, motor function and cognitive and neuropsychiatric functions in G2019S carriers from the whole series of 1245, in order to achieve a full clinical characterization of patients with this mutation. 2. Methods 2.1. Subjects We studied 1245 unrelated consecutive patients with degenerative parkinsonism, who contributed to the DNA Bank of the Parkinson Institute, Istituti Clinici di Perfezionamento, Milan ( Human genetic Bank of patients affected by Parkinson disease and parkinsonisms ; A first group of 629 patients was tested for the following major LRRK2 mutations: C4321 T (R1441C), C4321G (R1441G), A5096G (Y1699C), and G6055A (G2019S). The frequencies of the mutations in this group have been reported previously [20]. A second group of 616 samples was tested later for the G6055A (G2019S) mutation and the results are reported here for the first time. Patients were those attending the Parkinson Institute, who participated to the DNA Bank initiative, regardless of family history of PD or age at onset of the disease. In addition, if the G2019S mutation was identified in a proband, we studied all family members available with certain PD. In this way we enrolled five additional familial cases from four families. All 1245 patients studied had a diagnosis of primary degenerative parkinsonism. One thousand and ninetytwo fulfilled clinical criteria for PD, 14 dementia with Lewy bodies (DLB), five frontotemporal dementia (FTD), 42 multiple system atrophy (MSA), 31 progressive supranuclear palsy (PSP) and 18 corticobasal degeneration (CBD). In the remaining 43 cases the clinical diagnosis was still uncertain and these patients are here reported as undefined primary parkinsonism (PKS). The clinical diagnosis of PD was established according to the UK Parkinson s Disease Society Brain Bank criteria [21,22]. The diagnosis of PD required the presence of bradykinesia and at least one of the following: resting tremor, rigidity and postural instability; a positive response to dopaminergic therapy; the absence of atypical features or other causes of parkinsonism. In the non-pd cases we referred to the diagnostic criteria published by Litvan and colleagues [23]. The parkin gene was tested in all PD patients with onset p40 years of age. Eleven patients were previously found to carry parkin pathogenic mutations and were therefore excluded from this study. Among the 1092 PD patients 659 were male (60.3%), the mean age at onset was years (range 14 82; SD710.42), the mean disease duration was years (range 1 56; SD76.15). Except for 19 patients originating each from one of 19 different countries (Ukraine, Turkey, South Africa, Sri Lanka, USA, Principality of Monaco, Peru, Yugoslavia, Israel, Iceland, Ireland, Iran, United Kingdom, France, Ethiopia, Colombia, Argentina, Albania, Greece), all patients were of Caucasian ethnicity and Italian origin. The project was approved by the local Ethics Committee and written informed consent was obtained from all subjects Mutation analysis A first group of 629 patients was previously tested, using a SNaPshot method, for four pathogenic LRRK2 mutations: R1441C, R1441G, Y1699C, and G2019S; the genetic findings in this group have been reported previously [20]. A second group of 616 samples, including 463 PD and 153 atypical degenerative parkinsonisms, were tested only for the G6055A (G2019S) mutation using a previously described TaqMan allelic discrimination assay [24]; the genetic findings in these 616 samples are reported here for the first time Clinical studies of G2019S patients All patients with the G2019S mutation were examined by movement disorder neurologists. Unified Parkinson s Disease Rating Scale (UPDRS) [25] scores were noted

3 412 ARTICLE IN PRESS S. Goldwurm et al. / Parkinsonism and Related Disorders 12 (2006) both in On phase and in Off phase (after 12 h without drugs, whenever possible) together with Hoehn and Yahr scale [26]. The following clinical data were also collected for comparisons between G2019S carriers and non carriers: family history of PD, age at disease onset, asymmetry of symptoms at onset, disease duration, Hoehn and Yahr stage, UPDRS in On and Off state, period of levodopa therapy initiation. Patients were classified as familial if at least one relative among their first, second, or third degree relatives had a formal diagnosis of PD. The remaining patients were classified as sporadic. The age at which the patient noticed the first PD symptom was considered to be the age at onset of disease. Clinical diagnosis of dementia was made according to DSM-IV criteria Cognitive and neuropsychological assessment Seventeen G2019S carriers consented to be evaluated with a neuropsychological battery widely used for cognitive and neuropsychiatric assessment of PD patients. The battery included: Mini Mental State Examination MMSE [27] and Clock Drawing Task CDT [28] to assess general cognitive status; Rey Auditory Verbal Learning Test. RAVLT [29] to assess verbal learning and recall abilities; verbal fluencies (phonemic PF and category fluencies CF ) [30,31] to assess language production; Raven s Colored Progressive Matrices CPM [32,33], the Frontal Assessment Battery FAB [34,35], the Attentive Matrices [36] to assess frontal lobe functions. Neuropsychiatric status and behavior were evaluated with the Neuropsychiatric Inventory NPI [37] and Hamilton Depression Rating Scale HDRS [38]. Neuropsychological and neuropsychiatric assessments were always carried out in the On state Statistical analysis Values are given as mean 7SD. We measured differences between continuous variables with unpaired Student s t-test or Wilcoxon test according to the characteristics of data distribution. Differences in nominal variables between each group of patients were tested by chi-square test. A value of po0.05 was considered significant. Statistical analysis was carried out using JMP software of the SAS Institute. 3. Results 3.1. Prevalence of G2019S mutation Among the 616 patients reported here for the first time, the G2019S mutation was identified in heterozygous state in six subjects, all affected by PD. Considering the whole sample of 1245 patients (including those previously reported [20]), the G2019S mutation was identified in heterozygous state in 19 of the 1092 PD probands (1.74%; seven males and 12 females) and in none of the 153 probands with other degenerative parkinsonisms. No significant differences were observed in the distribution of the G2019S carriers according to age at onset (Table 1). Homozygous carriers of the G2019S mutation were not detected. Table 2 Distribution of the G2019S mutation according to family history of PD Patients G2019S (%) p a All Sporadic Familial p ¼ degree p ¼ At least one parent po Only sibling p ¼ degree p ¼ degree a Compared to sporadic patients. Table 1 Distribution of the G2019S mutation according to age at onset of the 1092 PD patients Age at onset Total p All cases G2019S (%) 4 (3.25%) 4 (1.52%) 7 (1.79%) 4 (1.28%) 19 (1.74%) Familial G2019S (%) 2 (7.14%) 3 (4.83%) 4 (4.54%) 1 (1.72%) 10 (4.24%) Sporadic G2019S (%) 2 (2.11%) 1 (0.50%) 3 (0.99%) 3 (1.18%) 9 (1.05%)

4 S. Goldwurm et al. / Parkinsonism and Related Disorders 12 (2006) The distribution of G2019S by family history is reported in detail in Table 2. The mutation was about 4 times more common in patients with a family history of PD than in sporadic cases (p ¼ ). Maximum frequency was recorded in patients with at least one parent suffering from PD (7.41%, po0.0001). The increase in frequency in patients with a family history of PD persisted when patients were subdivided into subgroups according to age at onset (Table 1) Clinical aspects: PD history and motor function Amongst the 19 G2019S carrier probands, four had living relatives with PD who took part in the study; among these secondary PD cases, four were men (two from the same family), and one was a woman. All five PD relatives carried the G2019S heterozygous mutation. Thus, we were able to characterize a total of 24 carriers of this mutation, including 11 males and 13 females. The main clinical features (PD history and motor function) of the 24 carriers are reported in Table 3. The comparison of some clinical features between G2019S carriers and noncarriers is reported in Table 4. Age at onset in the G2019S carriers varied considerably, ranging from 34 to 83 years. Mean onset age was 53 years, a value that was similar to the mean onset age of noncarriers (Table 4). Female G2019S carriers were on average 9 years younger at onset than male carriers (49.5 versus 58.9 years) and about 6 years younger than noncarriers female (55.2 years); the difference was not statistically significant (p ¼ 0.067, 0.08, respectively, Student s t-test). The duration of disease was similar in the various groups. Clinical motor features of G2019S carriers were those of idiopathic PD: tremor and bradykinesia were the most common symptoms at onset. In most cases (92%) the symptoms at onset were asymmetrical, as in noncarriers (88%). All G2019S carriers developed bradykinesia after 1 year and rigidity after 3 years of disease; tremor was absent in 8/24 (33%). H&Y stage and UPDRS scores (motor score and ADL) were similar in the two groups (Table 4). All carriers were treated with L-Dopa with good motor response. Levodopa therapy was initiated later in noncarriers compared to G2019S carriers (Table 4). About 70% of G2019S carriers developed long-term motor complications, such as dyskinesias and motor fluctuations, while about 40% developed tonic OFF dystonia, on average after 7 years of disease (Table 3). Three patients (817, 516, 789) underwent Deep Brain Stimulation (DBS) mainly due to severe dyskinesias, on average 9 years after the onset of disease. One patient, in whom onset of disease had occurred at the age of 43 years (317), underwent pallidotomy on the right side after 12 years (1987), and thalamotomy on the left side after 16 years (1991) for resting tremor and rigidity. Patient 1092 had onset of disease at the age of 35 years and developed severe cognitive decline at about 47 years of age, associated with apathy, depression and visual and auditory hallucinations throughout the day. Six carriers experienced hallucinations and intermittent delirium that partly remitted after therapy was reduced (Table 3) Clinical aspects: cognitive and neuropsychiatric assessments Seventeen subjects among the patients identified with G2019S mutation accepted to be assessed for their cognitive and neuropsychiatric status. A summary of the results is reported in Table 5. MMSE was mildly abnormal in two G2019S carriers and markedly abnormal in three, including the woman mentioned above (1092), who had overt dementia. Among the other four patients who had pathological MMSE scores, three presented frontal lobe dysfunction, and one had normal frontal lobe scores. Overall, frontal lobe deficits (as shown by FAB scores) were found in 35% of carriers (Table 5). According to Hamilton Depression Rating Scale scores, 69% of carriers were depressed. This finding is consistent with the NPI item 4 score, which suggests the presence of depression in a high proportion of carriers (50%) (Table 5). Interestingly, in five cases depressive symptoms developed at the same time of onset of PD motor symptoms and in one case (317) several years before the onset of PD motor symptoms. Anxiety and irritability were common among G2019S carriers (item 5: 62% and item 9: 56%). Also sleep disorders were common amongst carriers (item 11: 56%); they consisted mainly in repeated awakenings and insomnia. This finding is confirmed by the outcome of the neurological examination in 24 G2019S carriers (Table 3: sleep disorders 75%). Hallucinations were present in approximately onethird of the cases. NPI-12 score was altered (X12 points) in 14/16 (87.5%) of the carriers (Table 5). 4. Discussion We tested a large cohort of consecutive parkinsonian patients attending our centre for the most frequent LRRK2 mutation and analysed in detail the PD history and motor function. Moreover, we assessed for the first time the detailed neuropsychological and neuropsychiatric profile in G2019S mutation carriers. The main features of the PD history and motor function of the 24 carriers identified in our cohort of consecutive patients did not differ from those of

5 Table 3 Clinical features of the 24 G2019S carriers Total 1632 b Personal code 903 a 1092 a 516 a a 817 a 498 a 789 a a 1190 a b 794 a 499 b 869 a b a 902 a 1609 b Sex F F F M F F F M F M M F M M F M F F F M F M M M 11M/13F Positive family history for PD (63%) c Age at onset (years) , d Disease duration (years) , d Asymmetry at onset (92%) c Symptoms at onset Resting tremor (58%) c Bradykinesia (63%) c Rigidity (29%) c Postural instability (8%) c Gait impairment (8%) c Depression at onset (25%) c Clinical evolution Resting tremor (67%; 0,19) e Bradykinesia (100%; 0,67) e Rigidity (100%; 1,21) e Postural instability (75%; 5,44) e Gait impairment (96%; 3,83) e Freezing gait (79%; 7,79) e Sleep benefit (33%) c Sleep disorders (75%; 7,67) e Dysphonia (8%; 13,5) e Dysphagia (29%; 8,71) e Hoehn/Yahr Stage d UPDRS Motor score in On phase NA ,83710,17 d Motor score in Off phase 29 NA NA NA NA NA NA NA NA NA 36,87714,74 d ADL in On phase 2 NA NA NA NA ,5577,69 d ADL in Off phase 20 NA NA NA NA NA NA 12 NA NA 18,3878,82 d Therapy Levodopa latency (months) f d Levodopa daily dose (mg) , d Dopamine agonist daily dose (mg) g 5 2,9 4,5 1,4 0 25, ,9 4,5 2,9 4,29 2,86 1, ,86 4,3 0 1,5 2, ,5075,05 d Long-term levodopa syndrome Wearing off (75%; 6,24) e Dyskinesia (71%; 7,65) e Dystonia (41,67%; 6,8) e Cognitive-psychiatric symptoms h Depression anxiety (50%; 0,58) e Hallucination delusion (33%; 10) e Dementia + 1 (4%; 12) e S. Goldwurm et al. / Parkinsonism and Related Disorders 12 (2006) ARTICLE IN PRESS +presence of the symptoms; absence of the symptoms; NA ¼ not available. a Patients identified in the earlier study [20]. b Familial cases identified subsequently in the proband s family. c number of positive patients (% of positive patients). e number of positive patients (% of positive patients; mean years of presentation from the onset). d mean7standard deviation. f Time elapsed (months) from PD onset to initiation of levodopa therapy. g Dopamine agonist have been reported as Pergolide equivalent according to Grosset and colleagues [54]. h Cognitive-psychiatric symptoms reported in this table were collected by the neurologist at the clinical examination.

6 S. Goldwurm et al. / Parkinsonism and Related Disorders 12 (2006) idiopathic PD. Unilateral tremor and bradykinesia appeared at onset in most of the carriers, who all responded to levodopa and developed dyskinesia and/or motor fluctuations as the disease progressed. This comprehensive clinical picture in G2019S carriers is consistent with the SPECT analysis that showed similar decrements of [123I] Ioflupane tracer binding in carriers and noncarriers in our patient population [39]. Age at onset varied considerably, but was always within the range of idiopathic PD. Patients with the mutation were fairly evenly distributed amongst the various age categories, with a slight increase in patients with an early onset of disease (3.25% between p40 years versus 1.5% between 440 years). Female carriers had a tendency towards an earlier onset compared to male carriers and noncarriers. However, this has not been confirmed in other series [14]. The analysis of a larger number of carriers is necessary to verify this observation. The previously reported association of the mutation with longer disease duration and slower progression [9] was not observed in our study. Disease duration was similar to noncarriers, as well as Hoehn/Yahr stage and UPDRS evaluation, while Levodopa was used earlier in carriers. This might suggest a more aggressive disease course in carriers. Moreover, our series includes patients with severe PD that started early (o50 years) and developed motor complications requiring DBS. We found low MMSE values in 5/17 (29%) patients consistent with a reported prevalence of dementia in PD ranging between 17% and 31% [40]. This is higher than originally suggested in the first papers on LRRK2 [5,6]. However, since we did not perform a formal neuropsychological testing in our noncarrier patients, we cannot confirm the finding of worse MMSE scores reported in French PD G2019S carriers by Lesage and colleagues [41] when compared to noncarriers. Similarly, 35% of our cases presented frontal lobe abnormalities. The NPI-12 score indicated the presence of significant behavioural abnormalities in 14/16 carriers, mostly depression, anxiety, irritability, and hallucinations. We acknowledge that giving the lack of a control group we cannot claim that this frequency is significantly greater than what expected in PD. However, in an independent cohort of DBS candidates with similar disease duration we have reported mean NPI-12 scores of points [42]. This latter value is also consistent with literature values [43]. The presence of greater risk of behavioural abnormalities has also been reported in other forms of genetic PD [44]. We also found a high incidence of sleep disorders, in keeping with the findings reported by others in patients with other LRRK2 mutations [16]. The incidence of sleep disorders in the G2019S carriers was also higher than what is usually seen in PD [45]. We found the G2019S mutation in 1.74% of PD patients and in none of those with other forms of parkinsonism (though the latter group was much smaller). Notwithstanding a variety of clinical and pathological findings suggestive of atypical, non-lewybody parkinsonism in the first LRRK2-related familial cases [6], the results of the more recent studies and our own findings associate G2019S exclusively with typical PD [46 48]. However, most studies have targeted patients with classical PD, and therefore, the prevalence of G2019S and other LRRK2 mutations in the other forms of parkinsonism and dementia remains to be further explored. Our results confirm and extend our previous study, and indicate that the LRRK2 G2019S mutation is the most common known genetic determinant in PD patients in the Italian population [20]. Nearly 2% of PD patients carry this mutation, with the highest prevalence (4.25%) observed amongst familial cases. The mutation was also found in 1.05% of the sporadic PD cases. These frequencies are consistent with those Table 4 Comparison between G2019S carriers and noncarriers G2019S carriers Noncarriers p Mean SD n Mean SD n Age at onset (years) NS Disease duration (years) NS Hoehn/Yahr Stage NS UPDRS Motor score in On phase NS Motor score in Off phase NS ADL in On phase NS ADL in Off phase NS Levodopa Latency (months) a p ¼ NS: not statistically significant. a Time elapsed (months) from PD onset to initiation of levodopa therapy.

7 Table 5 Formal cognitive and neuropsychiatric assessment Total No. of positive (%) Mean (SD) Sex F F F M F F F M M F M M F F F M M 7M/10F Age at onset (years) (712.12) Disease duration (years) (76.04) Age at examination (years) (712.38) Education (years) (75.33) Cognitive assessment MMSE /17 (29%) (74.87) CDT NA NA NA + 1/14 (7%) 3.5 (70.85) RAVLT learning + 1/17 (6%) (710.78) RAVLT recall + 1/16 (6%) 7.89 (73.03) PF + 1/17 (6%) (79.92) CF + + 2/17 (12%) (710.99) CPM + + 2/17 (12%) 28.2 (75.69) FAB /17 (35%) (74,66) Att Matr NA + NA 2/16 (13%) (713.8) Psychiatric assessment HDRS + NA /16 (69%) (75,9) NPI item 1 delusion 0 NA /16 (6.25%) 1(73.09) NPI item 2 hallucination 6 NA /16 (31.25%) 2.37 (73.79) NPI item 3 agitation 2 NA /16 (18.75%) 2.25 (73.74) NPI item 4 depression 0 NA /16 (50%) 4.31 (72.62) NPI item 5 anxiety 6 NA /16 (62.5%) 5.62 (74.37) NPI item 6 euphoria 1 NA /16 (6.25%) 0.44 (71.5) NPI item 7 apathy 0 NA /16 (0%) 0.25 (71) NPI item 8 disinibition 0 NA /16(6.25%) 0.94 (72.41) NPI item 9 irritability 6 NA /16 (56.25) 4.94 (73.92) NPI item 10 motor behav 0 NA /16 (0%) 0.31 (71.01) NPI item 11 sleep 8 NA /16 (56.25%) 5.12 (74.14) NPI item 12 appetite dist 0 NA /16 (0%) 0.12 (70.5) NPI-10 (1 10) 21 NA /16 (81.2%) (712.33) NPI-12 (1 12) 22 NA /16 (87.5%) (713.06) + means a pathological value; means a normal value. NA ¼ not available. MMSE Mini Mental State Examination: valueso24 were considered pathological. CDT Clock Drawing Task: valueso2 were considered pathological. RAVLT Rey Auditory Verbal Learning Test; list learning: values o28.52 were considered pathological; list recall valueso4.68 were considered pathological. PF phonemic verbal fluencies: valueso16 were considered pathological. CF category fluencies: valueso24 were considered pathological. CPM-Raven s Colored Progressive Matrices: valueso17.5 were considered pathological. FAB Frontal Assessment Battery: valueso13.5 were considered pathological. Att Matr Attentive Matrices: valueso30 were considered pathological. HDRS Hamilton Depression Rating Scale : valueso12 were considered pathological. NPI Neuropsychiatric Inventory: 1 delusions, 2 hallucinations, 3 agitation/aggression, 4 depression, 5 anxiety, 6 euphoria, 7 apathy/indifference, 8 disinhibition, 9 irritability/liability, 10 aberrant motor behaviour, 11 sleep disturbances, and 12 appetite disturbances. For each item values46 were considered pathological. For NPI-10 (items 1 10) values X10 were considered pathological; NPI- 12 (items 1 12) values X 12 were considered pathological. S. Goldwurm et al. / Parkinsonism and Related Disorders 12 (2006) ARTICLE IN PRESS

8 S. Goldwurm et al. / Parkinsonism and Related Disorders 12 (2006) reported in a large series in North America [14]. When probands from families with a potentially dominant transmission (at least one parent or at least one second degree relative) were taken into consideration, the frequency of the mutation was about 5.77% (9/156), clearly higher than in families with only affected siblings, where its frequency was similar to that in sporadic PD (1.61% versus 1.05%, respectively). The G2019S mutation is associated with a specific haplotype present in all subjects, suggesting that the mutation was transmitted by a single ancient founder [11,17,20]. Recently, two article showed a surprisingly high frequency of G2019S mutation in North African Arabs and Ashkenazi Jews. Lesage and colleagues [49] studied 104 probands among North African Arabs showing that 37% of Familial PD and 41% of sporadic PD were G2019S carriers. Also two controls out of 151 were carriers. Among 120 Ashkenazi Jewish PD patients from New York City, Ozelius and colleagues identified the G2019S mutation in 29.7% of familial PD and in 13.3% of sporadic PD, while in the controls the mutation was present in 1.3% of subjects [50]. This suggests that the common founder originated from North Africa, where probably Ashkenazim originated [50], and explain the presence of a North South gradient in Europe: the G2019S prevalence is intermediate in Italy (this study), Spain and Portugal [51,52] and lower in France, Germany, and Poland [16,41,53]. Our patients were enrolled consecutively and were not selected according to clinical or familial features. Consequently, the G2019S frequency reported in this study should be an accurate estimate of the G2019S prevalence among Italian PD patients, at least within hospital-based cohorts. However, the G2019S prevalence in the PD from the general population might be different and remains to be explored in populationbased samples. It is difficult to accurately estimate the penetrance of the G2019S mutation. In our series many cases were sporadic (9/19). In previous studies the penetrance was assessed in selected familial cases; consequently, their findings are presumably higher on account of the presence of selection biases [11,17]. It is essential to carry out penetrance studies in consecutive series such as the one described in this paper. Study of the penetrance in our cohort is in progress. In conclusion, we confirm the genetic findings of our previous study [20] in a second large sample of 616 PD cases, and we provide analysis of the G2019S mutation frequency in our entire sample of 1245 unrelated PD cases. Clinical studies showed that the LRRK2 G2019S mutation is associated with the typical motor signs and symptoms of idiopathic PD. However, behavioural abnormalities were frequent, suggesting the presence of a greater involvement of the limbic system in mutation carriers. Additional prospective studies are warranted to confirm this hypothesis. Acknowledgements We thank all the patients and their relatives for their contribution. The DNA samples were from the Human genetic bank of patients affected by Parkinson disease and parkinsonisms of the Parkinson Institute Istituti Clinici di Perfezionamento. This DNA Bank is supported by the Italian Telethon Foundation (Grant no. GTF04007). This study was supported by grants from the Internationaal Parkinson Fonds (The Netherlands) to V. Bonifati and from the Fondazione Grigioni per il Morbo di Parkinson (award Maria Vincenzina Grosso ) to S. Goldwurm. We thank Jennifer Hartwig, MD, for help in preparing the manuscript. References [1] Eriksen JL, Wszolek Z, Petrucelli L. Molecular pathogenesis of Parkinson disease. Arch Neurol 2005;62: [2] Bonifati V, Oostra BA, Heutink P. Unraveling the pathogenesis of Parkinson s disease the contribution of monogenic forms. Cell Mol Life Sci 2004;61: [3] Gasser T. 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