DOI: /01.wnl d8. This information is current as of October 29, 2007

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1 The LRRK2 G2019S mutation in Ashkenazi Jews with Parkinson disease: Is there a gender effect? A. Orr-Urtreger, C. Shifrin, U. Rozovski, S. Rosner, D. Bercovich, T. Gurevich, H. Yagev-More, A. Bar-Shira and N. Giladi Neurology 2007;69; DOI: /01.wnl d8 This information is current as of October 29, 2007 The online version of this article, along with updated information and services, is located on the World Wide Web at: Neurology is the official journal of the American Academy of Neurology. Published continuously since 1951, it is now a weekly with 48 issues per year. Copyright 2007 by AAN Enterprises, Inc. All rights reserved. Print ISSN: Online ISSN: X.

2 The LRRK2 G2019S mutation in Ashkenazi Jews with Parkinson disease Is there a gender effect? A. Orr-Urtreger, MD, PhD C. Shifrin, MSc U. Rozovski, MD S. Rosner, MD D. Bercovich, PhD T. Gurevich, MD H. Yagev-More, PhD A. Bar-Shira, PhD N. Giladi, MD Address correspondence and reprint requests to Dr. Avi Orr-Urtreger, Director, Genetic Institute, Tel Aviv Sourasky Medical Center, 6 Weizmann Street, Tel Aviv 64239, Israel aviorr@tasmc.health.gov.il ABSTRACT Background: Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common genetic determinant of Parkinson disease (PD) identified to date, and have been implicated in both familial and sporadic forms of the disease. The G2019S change in LRRK2 exon 41 has been associated with disease at varying frequencies in Asian, European, North American, and North African populations, and is particularly prevalent among Ashkenazi Jews. Methods: We assessed the occurrence of the LRRK2 G2019S, I2012T, I2020T, and R1441G/ C/H mutations in our cohort of Jewish Israeli patients with PD, and determined the LRRK2 haplotypes in 76 G2019S-carriers detected and in 50 noncarrier Ashkenazi patients, using six microsatellite markers that span the entire gene. Results: Only the G2019S mutation was identified among our patients with PD, 14.8% in the Ashkenazi and 2.7% in the non-ashkenazi patients, and in 26% and 10.6% of the Ashkenazi familial and apparently sporadic cases. The carrier frequencies in the Ashkenazi and non- Ashkenazi control samples were 2.4% and 0.4%. A common shared haplotype was detected in all non-ashkenazi and half-ashkenazi carriers and in all full-ashkenazi carriers tested, except two. Women and patients with a positive family history of PD were significantly over-represented among the G2019S mutation carriers. Age at disease onset was similar in carriers and noncarriers. Conclusions: Our data suggest that the LRRK2 G2019S mutation plays an important role in the causality of familial and sporadic Parkinson disease (PD) in Israel and that gender affects its frequency among patients. Although testing symptomatic patients may help establish the diagnosis of PD, the value of screening asymptomatic individuals remains questionable until the penetrance and age-dependent risk of this mutation are more accurately assessed, and specific disease prevention or modifying interventions become available. Neurology 2007;69: GLOSSARY LRRK2 leucine-rich repeat kinase 2; PD Parkinson disease. Parkinson disease (PD) is the second most common neurodegenerative disorder, affecting approximately 1% of the population over the age of 65 years, increasing to 4 to 5% of the population by the age of 85 years. 1 While generally considered a sporadic disease, in the past decade, causal mutations in the -synuclein, PARKN, PINK1, and DJ1 genes have been identified in familial forms of parkinsonism, supporting a major role for genetic factors in the etiopathogenesis of PD. 2,3 Recently, pathogenic mutations in the leucinerich repeat kinase 2 (LRRK2) gene at the PARK8 locus (chromosome 12p11.2 q13.1) were identified in families with autosomal dominant late-onset PD. 4,5 LRRK2 is a 51 exon gene, encoding a 2,527 amino acid protein, LRRK2/dardarin. 4 This protein belongs to the ROCO group within the Ras/GTPase superfamily, character- From The Genetic Institute (A.O.-U., C.S., U.R., S.R., D.B., H.Y.-M., A.B.-S.) and Movement Disorders Unit, Parkinson Center, Department of Neurology (C.S., T.G., N.G.), Tel-Aviv Sourasky Medical Center; and The Sackler Faculty of Medicine (A.O.-U., C.S., T.G., N.G.), Tel- Aviv University, Tel-Aviv, Israel. Supported by grants from M.K. Humanitarian Fund, National Parkinson Foundation, Miami, FL, Tel Aviv Sourasky Medical Center Grant of Excellence, and Wolfson Foundation. Disclosure: The authors report no conflicts of interest. Copyright 2007 by AAN Enterprises, Inc. 1595

3 ized by the presence of several conserved domains: a Roc (Ras in complex proteins) and a COR (C-terminal of Roc) domain, together with a leucine-rich repeat region, a WD40 domain, and a protein kinase catalytic domain. 6 Point mutations have been found in patients with PD in almost all of the identified LRRK2 domains. The distribution of mutations along this gene, together with the lack of known deletions or truncations, and the dominant pattern of inheritance, are consistent with a gain-offunction mechanism. 7 The most frequent and well-investigated LRRK2 mutation, 6055G A in exon 41, encodes a G2019S change in the highly conserved activation loop of the kinase domain. 8 LRRK2 G2019S accounts for about 5 to 6% of familial and approximately 1 to 2% of apparently sporadic PD in European and North American populations, 9-14 and despite a wide spectrum of ethnic backgrounds, carriers of this mutation share common haplotypes, suggestive of a singlefounder effect 12,15,16 or two-founder events. 17 The significantly increased prevalence of the LRRK2 G2019S mutation recently reported in North African Arabs (37% in patients with familial PD, 41% in patients with sporadic PD, and 1% in controls) 18,19 and in North American Ashkenazi Jews (29.7% and 26.1% in patients with familial PD, 13.3% and 7.7% in patients with apparently sporadic PD, and 1.3% and 2.0% in controls), 20,21 sharing the previously reported common haplotype, suggested that this mutation originated in the Middle East. 20 We present the analysis of a large Jewish Israeli cohort of patients with PD and controls, including the largest group of Ashkenazi patients analyzed to date. The exact characterization of parental origins of all patients and control individuals allowed us to determine the frequency of the common G2019S LRRK2 mutations among the different Jewish populations. This mutation was detected mainly in patients with PD of Ashkenazi origin, but not in Jewish patients who originated from Iraq or Morocco. Further comparison of specific epidemiologic and clinical features between patient carriers of the LRRK2 G2019S mutation and noncarriers demonstrated that this mutation is significantly over-represented in Ashkenazi women with PD. METHODS Population. The study population included 472 Jewish patients, 35.8% women (n 169) and 64.2% men (n 303), treated at the Movement Disorders Unit, Tel-Aviv Sourasky Medical Center. All patients carried a diagnosis of clinically definite PD made by a movement disorder specialist, according to UK PD Society Brain Bank criteria. 22 Patients were enrolled consecutively and were not selected for any clinical or familial features. The recruitment interval spanned 11 months, from September 2005 to August All patients underwent a detailed interview to disclose ancestry, family history for PD or other movement disorders, age at disease symptom onset and diagnosis, and environmental and occupational risk factors. Patients with clinically significant dementia were excluded. Our cohort of Jewish patients with PD was not previously tested for mutations in PRKN, PINK1, and DJ1 genes. Ancestry and country of origin of both parents was reported by each participant. A total of 72.9% of the 472 Jewish patients (n 344) reported Ashkenazi ancestry on both paternal and maternal sides (table 1), in 23.7% both parents were non-ashkenazi (n 112), 3.0% of the patients were of mixed (half Ashkenazi) origin (n 14), and in 2 patients ancestry was unknown. Familial PD, defined as having at least one first- or second-degree relative with a diagnosis of PD, was noted in 27.9% of patients (131/469) with information regarding family history, 16.8% (79/469) of which had an affected first-degree relative. Mean age at symptom onset was years (SD 11.6, range 21 to 94), with early onset disease ( 50 years) reported by 24.6% (114/464) of the patients. The mean age at diagnosis was 60.3 years (SD 11.2, range 26 to 94) and the mean age at enrollment to this study was 67.6 years (SD 10.2; range 36 to 97). A total of 1,802 control samples were analyzed. These included 1,425 anonymous young Jewish individuals, aged 20 to 45, mostly women (500 of Ashkenazi origin, 400 of Moroccan origin, 300 of Iraqi origin, 100 of Turkish origin, and 125 of Bulgarian origin, table 1), who were randomly sampled. An additional 377 samples (341 Ashkenazi, 32 non Ashkenazi, and 4 mixed Ashkenazi, table 1) were collected from individuals aged 40 to 89 years, including 163 anonymous DNA samples from healthy Ashkenazi Jews, purchased from the National Laboratory for the Genetics of Israeli Populations, and 214 samples, mainly from Jewish spouses of patients with PD. In order to generate an elderly Ashkenazi control group of age- and sex-matched individuals, 252 subjects were randomly selected out of the 341 Ashkenazi controls (56.3% men, 43.7% women, mean age of years). Neurologic examinations were not performed on controls although, upon medical interview, individuals were self-reported as healthy and any spouse controls reporting a 1596 Neurology Downloaded 69 October from 16, 2007 at SKANFO INC on October 29, 2007

4 Table 1 Study group LRRK2 G2019S mutation carrier frequencies in Ashkenazi and non- Ashkenazi patients with Parkinson disease (PD) and controls No. tested No. G2019S carriers (%) Total PD patients (12.3)* Ashkenazi (14.8) Non-Ashkenazi Jews (2.7) Mixed (half Ashkenazi origin) 14 4 (28.6) Unknown origin 2 0 Total controls 1,802 Total Ashkenazi (2.4) Individuals age (2.6)* Young (age 20 45) (2.2) Total non-ashkenazi Jews (0.4) Individuals age * Total young individuals (age 20 45) (0.4) Morocco (0.25) Iraq Bulgaria (1.6) Turkey (1) Mixed (half Ashkenazi origin) Individuals age * *All Parkinson patients vs 377 Ashkenazi and non-ashkenazi controls: OR, 5.7 (95% CI 2.8 to 11.7, p ). Ashkenazi Parkinson patients vs total Ashkenazi controls: OR, 7.1 (95% CI 4.2 to 12.2, p 0.001). tremor of any kind, balance problems, or movement or gait disturbances were excluded from the study. Family pedigrees were constructed for all patients and spouse controls. None were from consanguineous families. Any related individuals (patients or controls) were excluded from the study. Structured family history interviews were not performed, however, two investigators obtained pedigree information together on the first 50 interviews. All patients and spouse controls signed an informed consent prior to entering this study. All DNA samples were coded and tested in an anonymous manner. The Institutional and National Supreme Helsinki Committees for Genetic Studies approved the study protocols and the informed consents. Mutation detection. Genomic DNA was isolated from peripheral blood using standard protocols or saliva according to manufacturer s instructions (Oragene, Ottawa, Canada). To detect the 6055G A (G2019S) and the nearby 6035T C (I2012T) and 6059T C (I2020T) mutations in LRRK2 exon 41, we amplified a 171 bp fragment with the following primers: forward 5= CCTGTGCATTTTCTG- GCAGATA 3= and reverse 5= CCTCTGATGTTTTTATC- CCCATTC 3=. 20 PCR reactions were performed using Sigma Taq Polymerase according to manufacturer s instructions (Sigma-Aldrich, St Louis, MO) using a Biometra PCR system (Biometra GmbH, Gottingen, Germany). DNA alterations were analyzed using a WAVE DHPLC apparatus (Transgenomic Inc., Omaha, NE), as described previously. 23 Heteroduplexes were detected at 60.4 C. Mixing DHPLC studies using normal DNA were performed on all patient and Ashkenazi control samples in order to detect homozygote carriers. All samples with variant heterochromatograms were sequenced using the BigDye Terminator Chemistry (Applied Biosystems, Foster City, CA) and analyzed using an automated ABI Prism 310 Genetic Analyzer (Applied Biosystems). DHPLC screening for the 4321C G, 4321C T, and 4322G A (R1441G, R1441C, and R1441H) mutations in LRRK2 exon 31 was performed using the following primers: forward 5= GCAAATATCAA- CAGGAATGT 3= and reverse 5=ACTTCTCAAAATT- TCTGACA 3=. 24 Haplotype analysis. The LRRK2 haplotype was determined for 76 G2019S carriers (58 patients with PD and 18 controls) and for 50 noncarrier Ashkenazi patients with PD by genotyping six microsatellite markers, D12S2514, D12S2515, D12S2516, D12S2518, D12S2519, and D12S2520, as described previously. 12 The length of each amplified product was determined using ABI Prism 310 Genetic Analyzer and analyzed using GeneScan Analysis Software version (Applied Biosystems). Haplotype analysis was performed using HPLUS (V 2.5) software. This program accepts various types of biomarkers, such as microsatellites, and estimates the frequency of the shared haplotype in association studies in the cases vs the control population using a valid statistical model. 25 Statistical analysis. Comparison of demographic and clinical characteristics between mutation carriers and noncarriers among total PD patient population and among Ashkenazi patients only was performed. Categorical variables were tested using 2 or Fisher exact test, and continuous variables were tested by Student t test. Medians were compared using the Mann-Whitney test. OR for all mutation carriers was calculated using logistic regression model with an estimated 95% CI. All of the above analyses were done using SPSS V.13 (SPSS Inc., Chicago, IL). OR was also calculated for Ashkenazi mutation carriers only, using online computation software (DJR Hutchon Calculator). RESULTS The LRRK2 G2019S mutation is frequent in Ashkenazi Jewish patients with PD. Out of all Jewish patients with PD tested, 12.3% (n 58) carried the LRRK2 G2019S mutation in a heterozygous state. The carrier frequency among patients with PD of Ashkenazi origin on both parental sides was 14.8% and 2.7% among non- Ashkenazis (2 of Bulgarian and 1 of Syrian origins) ( , df 2, p 0.001) (table 1). None of the patients with PD originating from Iraq (n 19) or Morocco (n 23, 2 of them of mixed Moroccan origin) carried this mutation. Among the small group of patients with mixed Ashkenazi origin, a relatively high percentage were mutation carriers (28.6%). Estimations of OR for a mutation carrier to develop PD was 5.7 (95% CI 2.8 to 11.7, p ) when comparing the entire patient cohort to the 377 Ashkenazi and non-ashkenazi controls Neurology 69 October 16,

5 (table 1). Notably, the G2019S mutation was detected in 5 of the 252 (2%) Ashkenazi age- and sex-matched control samples, and the OR for an Ashkenazi mutation carrier to develop PD was 8.6 (95% CI, 3.4 to 21.9, p ) when comparing the 344 Ashkenazi patients with PD to the 252 age- and sex-matched elderly controls. Furthermore, since possible mutations in the recessively inherited PD-related genes, PRKN, PINK1, and DJ1, which are associated with early onset disease, may mask the results in the founder Ashkenazi Jewish population, an additional association analysis was performed, comparing 254 late-onset ( 50 years) Ashkenazi patients with PD and 318 Ashkenazi controls over the age of 50 years. The G2019S carrier frequencies in these two groups were 13.8% (35/254) and 2.5% (8/318), yielding an OR of 6.2 (95% CI, 2.8 to 13.6, p ). In order to assess the frequency of the G2019S mutation in the general Jewish Israeli population, we sampled 1,425 young healthy controls. Similar to the prevalence of mutation carriers found among the 252 Ashkenazis matched by age and sex (2%), 2.2% of the young Ashkenazis carried the G2019S mutation, while only 0.4% of the young non-ashkenazis were G2019S carriers. No carriers were detected in the young controls of Iraqi origin. Homozygous carriers of the G2019S mutation were not detected in any of the 2274 DNA samples tested from patients and controls. Neither the 6059T C, I2020T missense mutation, affecting the adjacent codon (previously identified in a German kindred 4 and in the original PARK8-linked Japanese Sagamihara kindred 26 ), nor the 6035T C, I2012T missense mutation (previously identified in a Taiwanese PD patient 27 ) were detected in any of the 2,274 patients and control individuals tested. The three previously identified mutations at position 1441 in exon 31 of LRRK2, R1441C, 4 R1441G, 5 and R1441H 28 were not identified in any of the 472 patients with PD. Finally, two silent sequence variations were detected, 6054 C T (Y2018Y) in 3 Ashkenazi control individuals and 6013 C T (L2005L) in one control of Moroccan origin. Allele frequency and haplotype analysis. Partial haplotype analysis using six microsatellite markers that span 240 Kb encompassing the entire LRRK2 gene was performed in the 76 G2019S carriers and in 50 noncarrier Ashkenazi patients with PD as detailed in table 2. A shared haplotype, D12S2514(291)-D12S2515(224)-D12S2516(254)- D12S2518(154), consistent with that described previously, 12,15,16 was revealed in 97% of carriers tested. Two Ashkenazi carriers (3%), one patient Table 2 Marker Allele frequency and haplotype analysis: Allele frequencies of six chromosome 12q12 markers in 76 LRRK2 G2019S carriers of Ashkenazi and non-ashkenazi origin and in 50 Ashkenazi noncarrier patients with Parkinson disease No. of alleles (%) In carrier, n 152 In noncarrier, n 100 Alleles: Sizes in base pairs No. % No. % D12S * 106* 69.7* 45* 45.0* D12S * 24.0* 224* 86* 56.6* D12S * 127* 83.6* 67* 67.0* D12S * 135* 88.8* 84* 84.0* D12S * 95* 62.5* 36* 36.0* D12S * 51.0* 260* 77* 50.7* *The most frequent allele in each marker. and one control, shared a less frequent haplotype D12S2514(291)-D12S2515(216)-D12S2516(254)- D12S2518(154) (table 3). Using HPLUS software, we calculated that the most frequent shared haplotype was D12S2514(291)-D12S2515(224)- D12S2516(254)-D12S2518(154)-D12S2519(132)- D12S2520(260). This extended haplotype was detected in 26.5% of mutation carrier compared to 2.0% in noncarrier individuals. Of note, this extended shared haplotype was detected in all four carrier individuals who reported mixed Neurology Downloaded 69 October from 16, 2007 at SKANFO INC on October 29, 2007

6 Table 3 Allele frequency and haplotype analysis: Presence of the shared alleles among Ashkenazi and non- Ashkenazi LRRK2 G2019S carriers and in 50 Ashkenazi noncarrier patients with Parkinson disease Carriers Marker Ashkenazi, n 65 Mixed- and non-ashkenazi origin, n 11 Total, n 76 Noncarriers, Ashkenazi patients, n 50 Size of shared allele (bp) No. % No. % No. % No. % D12S D12S D12S D12S D12S D12S Ashkenazi origin (only one parent of Ashkenazi origin) and in all seven non-ashkenazi carrier patients and controls. The LRRK2 G2019S mutation is significantly more frequent in two groups of patients with PD: Women and familial cases. Table 4 presents the demographic and clinical characteristics and the comparisons between the 58 patients heterozygous for the G2019S mutation and all 414 noncarrier patients, and between the 51 Ashkenazi carrier patients and the 293 Ashkenazi noncarrier patients. The 58 LRRK2 G2019S carriers included 16.6% Table 4 Comparisons between LRRK2 G2019S carriers and noncarriers in all patients with Parkinson disease and in patients of full Ashkenazi origin Total patient population Ashkenazi patients Mutation carriers (58) Noncarriers (414) p Value Mutation carriers (51) Noncarriers (293) p Value Ashkenazi 87.9 (51) 70.8 (293) Non-Ashkenazi 5.2 (3) 26.3 (109) Mixed Ashkenazi 6.9 (4) 2.4 (10) Unknown 0 (0) 0.5 (2) Women 48.3 (28) 34.1 (141) 0.034* 51.0 (26) 34.8 (102) 0.027* Age at symptoms onset, y (SD) 56.9 (11.9) 59.4 (11.6) (11.6) 59.2 (11.6) 0.38 Early onset 50 y 30.4 (17) 23.8 (97) (14) 24.5 (71) 0.54 Family history: PD in first-degree relative Family history: PD in firstor second-degree relative 32.8 (19) 14.6 (60) 0.001* 33.3 (17) 14.4** (42) 0.001* 48.3 (28) 25.1 (103) * 49.0 (25) 24.4** (71) * Apparently sporadic 51.7 (30) 74.9 (308) * 51.0 (26) 75.6** (220) * Presenting symptoms/signs Resting tremor 60.3 (35) 56.5 (234) (32) 56.0 (164) 0.44 Bradykinesia 17.2 (10) 18.6 (77) (9) 20.5 (60) 0.71 Rigidity 34.5 (20) 28.7 (119) (16) 27.6 (81) 0.61 Gait difficulty 19.0 (11) 19.3 (80) (8) 20.5 (60) 0.57 Postural instability 12.1 (7) 7.5 (31) (7) 6.8 (20) 0.10 Micrographia 1.7 (1) 8.7 (36) (26) 0.020* All data are presented as percentages (number of patients) except for number of years (SD). *p Values are significant. Information was available for 56 carrier patients. Information was available for 408 noncarrier patients. Information was available for 49 Ashkenazi carrier patients. Information was available for 290 Ashkenazi noncarrier patients. Information was available for 411 noncarrier patients. **Information was available for 291 Ashkenazi noncarrier patients. Neurology 69 October 16,

7 of the women with PD (28/169) and 9.9% of the male patients (30/303). Women with PD were significantly over-represented among carriers, relative to their representation in the entire PD cohort. Out of the PD patient carriers, 48.3% (28/ 58) were women compared to 34.1% (141/414) women among the noncarrier patients ( , df 1, p 0.034). This difference was also significant when analyzing Ashkenazi patients separately. While 51% (26/51) of the Ashkenazi mutation carriers were women, only 34.8% (102/ 293) of Ashkenazi noncarrier patients were women ( , df 1, p 0.027). This result should be interpreted cautiously, since when applying the Bonferroni correction for multiple comparisons, which sets the p value from the a priori 0.05 to , the gender difference is only of borderline significance. Logistic regression model using the demographic characteristics as covariate (age at onset, family history, smoking status, and gender) found gender [Exp( ) 2.1 (CI 1.2 to 3.8)] and family history of a first degree relative [Exp( ) 2.5 (CI 1.4 to 4.4)] to be significant predictors of the mutation status, further substantiating the significant over-representation of women among LRRK2 G2019S mutation carriers. In addition, covariate analysis controlling for gender did not reveal any other significant variables, except family history, that differentiated between mutation carriers and noncarriers. The LRRK2 G2019S mutation was detected in both familial and sporadic patients. However, it was significantly more frequent in cases with positive family history. While 32.8% of the mutation carriers had a first-degree relative with PD, only 14.6% of the noncarriers had affected first-degree relatives ( , df 1, p 0.001). Similarly, 48.3% of the mutation carrier patients had either first- or second-degree relative with PD, while only 25.1% of the noncarrier patients had a positive family history of PD ( , df 1, p ). A similar analysis that included only patients of Ashkenazi origin from both parental sides revealed that 33.3% and 49.0% of mutation carriers had first-degree or either first- or seconddegree relatives with PD, while only 14.4% and 24.4% of the noncarriers had first-degree or either first- or second-degree relatives with PD ( , df 1, p 0.001, and , df 1, p ). Additionally, we determined the frequency of the G2019S mutation among our 344 Ashkenazi patients with PD, with and without family history of PD. The Ashkenazi PD patient carriers included 26% (25/96) of the familial cases and 10.6% (26/246) of the apparently sporadic cases. No major differences in the natural history and clinical features were noted between G2019S mutation carrier and noncarrier patients. The mean age at symptom onset in the 58 G2019S mutation carriers was years, compared to years in noncarriers (p 0.131). Age at symptom onset in mutation carriers was not gender related (women years, men , p 0.7). The clinical presentation was similar among mutation carriers and noncarriers, with rest tremor, rigidity, and gait difficulty as the most frequent presenting symptoms in both groups (table 4). To exclude the possibility of ascertainment bias, the epidemiologic and clinical variables were also compared between the 293 noncarrier Ashkenazi and 109 noncarrier non-ashkenazi patients, not including patients with unknown or mixed (half Ashkenazi) origin. These two groups of noncarrier patients were similar in all parameters tested. In addition, when comparing the 51 Ashkenazi LRRK2 G2019S carriers to the 293 Ashkenazi noncarriers, findings were similar to those in the analysis of the total patient population (table 4). DISCUSSION We have detected the LRRK2 G2019S mutation, possibly the most significant single genetic determinant of PD identified to date, in 14.8% and 2.4% of our Ashkenazi Jewish patients and controls. The Ashkenazi PD patient carriers of this mutation included 26% (25/96) of the familial cases and 10.6% (26/246) of the apparently sporadic cases, compared with carrier frequencies of 29.7% in familial and 13.3% in sporadic PD cases 20 and 26.1% in familial and 7.74% in sporadic PD cases 21 in two previous North American Ashkenazi cohorts. When combining our data with these two studies, the carrier frequency of the G2019S mutation was 26.9% among familial and 10.1% in apparently sporadic Ashkenazi PD cases, and 2.1% in healthy Ashkenazi controls (of note, the definition of familial cases differed among the three studies: one included first-degree relatives only, 21 another included first-, second-, and third-degree relatives, 20 while our data included first- and second-degree relatives). These frequencies were significantly higher than reported in different European and North American studies, 9-12 suggesting that the LRRK2 G2019S mutation plays a major role in the causality of PD in Ashkenazi Jews. Of note, the incomplete age-related penetrance of LRRK Neurology Downloaded 69 October from 16, 2007 at SKANFO INC on October 29, 2007

8 G2019S, with variable lifetime penetrance estimates ranging from 24 to 85% in different study populations, 12,20,21 may challenge the apparently sporadic nature of PD in LRRK2 G2019S carriers in a proportion of our patients, whose negative family history may in fact be the result of early death of family members, during World War II, before illness development. Interestingly, despite the significant association between G2019S and familial or sporadic PD in a study of North African Arabs (37 and 41%), a population that included Sephardic Jews, 18,19 none of the Jewish North African/Moroccan familial or sporadic PD patients in our cohort carried the G2019S change. In addition, of the 400 Jewish Moroccan controls, the G2019S mutation was detected in only one individual. Thus, our data suggest that this mutation is not frequent in North African/Moroccan Jews. The LRRK2 haplotype that was associated with the G2019S mutation in the non-ashkenazi and Ashkenazi of mixed-origin (half Ashkenazi) carriers tested and in all but two Ashkenazi carriers was consistent with that previously described. 12,15,16 A founder common to the G2019S carriers was initially suggested to have lived in the 13th century, 15 although more recently, was estimated to have lived 1,875 to 2,250 years ago, coinciding with the period of the Jewish diasporas. 17 Our partial haplotype analysis does not allow the calculation of the mutation age, however, the absence of patient and control mutation carriers of Iraqi origin further suggests that the LRRK2 G2019S mutation emerged only after the dispersion of the Jewish people in the Diaspora, circa 70 ad. 29 The similar presentation and a clinical course of PD in G2019S mutation carriers and noncarriers have been reported previously, 8,12-14,18,30,31 including homozygous LRRK2 G2019S mutation carrier patients who did not show earlier disease onset or distinctive clinical signs. 9,18,32 Since only mild tendency for earlier age at symptom onset was detected in G2019S carriers, our data also support a similar disease course in carriers and noncarriers. Notably, mutation carriers were more likely than noncarriers to be women (p 0.03), as recently observed in a North American cohort. 21 However, there were no gender-related differences between carrier men and women regarding family history or clinical features, and the OR for women G2019S carriers to have PD was 5.47 (95% CI 2.30 to 12.76) in our entire cohort and 5.94 (95% CI 2.49 to 14.18) when calculated for the Ashkenazi women only, similar to the ORs calculated for men and women together. Furthermore, the age at symptom onset in men and women carriers did not differ in our study, unlike the previously reported earlier disease onset in women G2019S carriers. 16 Since it is well documented that the overall frequency of PD is gender related, and is about 1.5 times higher in men than in women, 33 our data further suggest the existence of gender-related susceptibility factors that play important roles in PD development. Furthermore, it is also possible that some of these yet unknown gender effects may be specifically associated with LRRK2-related cellular pathways. The significant frequency of LRRK2 G2019Sassociated PD in Ashkenazi Jews will likely increase the demand for genetic testing by the patients and their family members, as well as by physicians. It is therefore important that the utility and consequences of both diagnostic and presymptomatic testing be considered carefully. Mutation status is particularly important when results have diagnostic or therapeutic consequences. In the case of PD, in individuals with a questionable extrapyramidal clinical picture, the detection of the LRRK2 G2019S mutation might support the diagnosis of PD. However, while the advantages of testing asymptomatic family members include close follow-up and early detection of PD, as well as the opportunity to plan both psychologically and financially for the future, these must be weighed against the negative emotional and social consequences, possible employment and insurance discrimination, and inconclusive counseling due to the partial penetrance and the inadequate understanding of the age-dependent risk of the LRRK2 G2019S mutation. 34 Therefore, as long as there is no proven medical or behavioral treatment that can modify the natural history of PD, there is currently no justification for clinical testing of asymptomatic family members. ACKNOWLEDGMENT The authors thank the patients and their spouses for their participation in this study. They also thank Ilana Elroi; Sonya Soloviov; Drs. Balash, Merims, and Shabtai; and Orna Moore for their assistance. Received October 24, Accepted in final form May 4, REFERENCES 1. Eriksen JL, Wszolek Z, Petrucelli L. Molecular pathogenesis of Parkinson disease. Arch Neurol 2005;62: Gasser T. Genetics of Parkinson s disease. Curr Opin Neurol 2005;18: Neurology 69 October 16,

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10 The LRRK2 G2019S mutation in Ashkenazi Jews with Parkinson disease: Is there a gender effect? A. Orr-Urtreger, C. Shifrin, U. Rozovski, S. Rosner, D. Bercovich, T. Gurevich, H. Yagev-More, A. Bar-Shira and N. Giladi Neurology 2007;69; DOI: /01.wnl d8 This information is current as of October 29, 2007 Updated Information & Services Subspecialty Collections Permissions & Licensing Reprints including high-resolution figures, can be found at: This article, along with others on similar topics, appears in the following collection(s): Parkinson's disease/parkinsonism nsonism All Clinical Neurology All epidemiology Risk factors in epidemiology ogy All Genetics Association studies in genetics netics Information about reproducing this article in parts (figures, tables) or in its entirety can be found online at: Information about ordering reprints can be found online: