ORIGINAL CONTRIBUTION. Screen for Excess FMR1 Premutation Alleles Among Males With Parkinsonism

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1 ORIGINAL CONTRIBUTION Screen for Excess FMR1 Premutation Alleles Among Males With Parkinsonism Jeremy Kraff, MD; Hiu-Tung Tang, BA; Roberto Cilia, MD; Margherita Canesi, MD; Gianni Pezzoli, MD; Stefano Goldwurm, MD, PhD; Paul J. Hagerman, MD, PhD; Flora Tassone, PhD Background: Individuals with fragile X associated tremor/ataxia syndrome frequently have associated features of parkinsonism, often leading to an initial diagnosis of Parkinson disease or other parkinsonism spectrum disorders. Parkinson disease populations may thus include individuals who harbor premutation expansions ( CGG repeats) of the fragile X mental retardation 1 (FMR1) gene. Objective: To screen DNA samples (male) from an Italian Parkinson disease clinic for an excess of premutation expansions of the FMR1 gene. Design: DNA samples obtained from 903 unrelated males through consecutive clinic visits were analyzed by an enhanced polymerase chain reaction method for detecting expanded CGG repeats Setting: Diagnostic assessments were performed at the Parkinson Institute, Istituti Clinici di Perfezionamento, Milan, Italy. Genotyping was conducted at the University of California Davis School of Medicine. Participants: A cohort of unrelated males with clinical features of parkinsonism. All but 12 males were of Italian origin, and all reported Caucasian ethnicity. Main Outcome Measure: CGG repeat number. Results: Three premutation carriers (61, 69, and 80 CGG repeats) were identified (0.33%), which is not significantly higher than the frequency of premutation alleles in the general population. The outcome of the current study, the largest screen of individuals with parkinsonism to date, supports previous screens of smaller parkinsonism cohorts. Conclusion: Broad screening for premutation alleles in Parkinson disease populations is unlikely to be productive in the absence of additional clinical or family history data that suggest involvement of the FMR1 gene. Arch Neurol. 2007;64(7): Author Affiliations: Department of Biochemistry and Molecular Medicine, School of Medicine, University of California, Davis (Drs Kraff, Hagerman, and Tassone); Medical Investigation of Neurodevelopmental Disorders Institute, University of California Davis Medical Center, Sacramento (Mr Tang and Drs Hagerman and Tassone); and Parkinson Institute, Istituti Clinici di Perfezionamento, Milan, Italy (Drs Cilia, Canesi, Pezzoli, and Goldwurm). FRAGILEX ASSOCIATED TREMOR/ ataxia syndrome (FXTAS) is a late-onset (age, 50 years), progressive neurological disorder with core features of action tremor and cerebellar gait ataxia. Frequent associated features include parkinsonism, executive dysfunction, and cognitive decline with features of frontal subcortical dementia, dysautonomia, and peripheral neuropathy. 1-5 Characteristic features on magnetic resonance imaging (MRI) include a high signal on T2 fluidattenuated inversion recovery imaging of the middle cerebellar peduncles (MCP sign), periventricular and subcortical white matter changes, and brain volume loss. 6,7 Neuropathology includes the presence of intranuclear inclusions in both astrocytes and neurons of clinically affected individuals. 8,9 Fragile X associated tremor/ataxia syndrome is caused by moderate expansions ( repeats [premutation range]) of a CGG trinucleotide in the fragile X mental retardation 1 (FMR1) gene. The pathogenesis of this disorder involves a direct toxic effect of overexpression of the FMR1 messenger ribonucleic acid. 10 Larger expansions ( 200 repeats [full mutation range]) generally accompanied by DNA methylation, transcriptional silencing, and the consequent absence of the fragile X mental retardation 1 protein (FMRP) give rise to fragile X syndrome, the leading heritable form of cognitive impairment (Online Mendelian Inheritance in Man [OMIM] *309550). In a recent retrospective study of the initial diagnoses of male FXTAS patients, 11 a quarter of the patients surveyed had received an initial diagnosis of some form of parkinsonism, suggesting that some carriers of premutation alleles may be found within cohorts of individuals who are being observed in Parkinson disease (PD) clin- 1002

2 ics. Initial surveys of several groups of patients with PD or parkinsonism failed to detect an excess of premutation allele. 12 However, the screened groups were relatively small, and some would not have detected even relatively high frequencies of premutation alleles within the samples. To further address this issue, we have screened DNA samples from a cohort of 903 males with clinical features of parkinsonism. This cohort is more than twice as large as any previously screened group. METHODS SUBJECTS A polymerase chain reaction based screen for premutation alleles of the FMR1 gene was performed on a cohort of 903 unrelated male patients who had received a diagnosis of either primary degenerative parkinsonism or essential tremor and who had contributed samples to the Human Genetic Bank of Patients Affected by Parkinson Disease and Parkinsonisms, Istituti Clinici di Perfezionamento, Milan, Italy ( /dnabank.html). The cohort was selected on the basis of consecutive clinic visits, regardless of family history of parkinsonism or age at onset of the disease. Written informed consent was obtained from all subjects. Patients fulfilled criteria for the following: 776 for idiopathic PD, 14 for dementia with Lewy bodies, 3 for frontotemporal dementia, 21 for multiple system atrophy, 21 for progressive supranuclear palsy, 9 for corticobasal degeneration, and 26 for essential tremor. For the remaining 33 cases, the clinical diagnosis was still uncertain; those patients are reported as having undefined primary parkinsonism. The clinical diagnosis of PD was established according to the UK Parkinson s Disease Society Brain Bank criteria, 13,14 which require the presence of bradykinesia, the absence of atypical features or other causes of parkinsonism, and at least 1 of the following: resting tremor, rigidity and postural instability, and a positive response to dopaminergic therapy. In the non-pd cases, diagnoses were assigned according to published diagnostic criteria. 15,16 Among the 776 patients with idiopathic PD, the mean age at onset was years (range, years; SD, years), and the mean disease duration was years (range, 1-56 years; SD, 6.3 years). All patients with PD were previously tested for the LRRK2 G2019S mutation and 10 were found to be carriers. 17 The parkin gene was tested in all PD patients with an age at onset of 40 years or younger and 8 were homozygous for the mutated gene ( J.K., unpublished data, 2007). Such patients were not excluded from this analysis. All but 12 patients were of Italian origin. The other patients were from Turkey, South Africa, Sri Lanka, the United States, Yugoslavia, Romania, Ireland, Iran, France, Albania, and Argentina. All patients reported Caucasian ethnicity. CASES IDENTIFIED AS PREMUTATION CARRIERS Case 1 (C-0230) is a 65-year-old man with an 18-year history of PD. The onset of symptoms occurred when he was aged 48 years, with bradykinesia and rigidity on the right side. He had a good response to treatment with dopaminergic agents. He first experienced wearing-off motor fluctuations 9 years after his initial presentation and peak-dose dyskinesias after 14 years. He progressively developed severe motor fluctuations and dyskinesia and was considered for subthalamic nucleus deep brain stimulation 14 years after initial symptoms. Neurological examination was assessed both 12 hours after last medication intake (off) and immediately after medication intake (on), with an improvement in Unified Parkinson Disease Rating Scale motor score of 29 to 15 ( 49%). Brain MRI showed mild corticosubcortical atrophy but no white matter signal abnormalities. Brain perfusion single-photon emission computed tomography showed bilateral striatal perfusion reduction. Neuropsychological evaluation revealed no specific cognitive deficit but disclosed psychotic symptoms, such as auditory hallucinations, jealousy, and persecution delusions. The neuropsychological follow-up assessment (1 year later) revealed mild difficulties with memory and frontal lobe related abilities but also showed improvement of psychiatric symptoms. Therefore, the patient underwent deep brain stimulation surgery and showed a significant clinical improvement, with reduction of daily time spent in off condition and dyskinesia. Hallucinations were not reported after surgery, but delusions occasionally occurred. Case 2 (B-0556) is a 50-year-old man with a 16-year history of PD. The onset of symptoms occurred at 34 years of age with resting tremor on the right hand and slowly progressive bradykinesia and rigidity. Dopaminergic treatment was instituted 6 years after the first symptoms appeared. He always showed a good clinical response to medication, experiencing wearing-off fluctuations 11 years from the onset, but no other motor complications. At48yearsofage, neuropsychologicalassessmentrevealedmild deficits of frontal lobe functions in the patient. Psychotic features, suchasjealousyandpersecutiondelusions, werereportedandpartially improved with quetiapine treatment. Brain imaging investigation results, such as MRI and perfusion single-photon emission computed tomography results, are not available. Case 3 (G-0444) is a 63-year-old man who first complained of micrography, bradykinesia, and rigidity on the right side at 43 years of age. The diagnosis of PD was established and dopaminergic treatment was started with good response. He developed motor fluctuations and dyskinesias 7 years following symptom onset. Clinical, radiological, and molecular findings are presented in Table 1. Neurological examination at 60 years was assessed both in off and on states, with an improvement in Unified Parkinson Disease Rating Scale motor score from 31 to 25 ( 20%). The neuropsychological tests detected mild frontal deficits characterized by reduction in attention and presence of preservative behavior. Impulsiveness, slight apathy, and depressed mood were also observed. Brain MRI showed moderate subcortical and cerebellar atrophy. Brain perfusion single-photon emission computed tomography revealed bilateral cortical hypoperfusion in the left posterior parietal and occipital areas and asymmetric blood flow reduction in the striatum bilaterally, mainly on the left side. MRI ASSESSMENT Following the identification of FMR1 premutation alleles, a neuroradiologist reexamined MRIs of cases 1 and 3 (patients C-0230 and G-0444). Cerebellar atrophy was noted in case 3. However, neither case demonstrated the high-signal lesions (T2- weighted) of the middle cerebellar peduncles (MCP sign) that are characteristic of FXTAS. 6 MOLECULAR MEASURES Genomic DNA was isolated from peripheral blood lymphocytes using standard phenol-chloroform extraction methods. DNA from each patient was amplified using an enhanced poly- 1003

3 Table 1. Clinical, Radiological, and Molecular Findings in PD Patient Carriers of the Fragile X Premutation Case Age at Onset, y Family History of Movement Disorders or Neurodegenerative Diseases Disease Duration, y Clinical Presentation 1 48 None 18 Bradykinesia, rigidity Levodopa Response Cerebellar Disorder Other Features Good, 45% None Psychosis (hallucination, delusion) 2 34 None 16 Resting tremor Good, 45% None Psychosis (delusions) 3 43 None 19 Bradykinesia, rigidity Abbreviations: NA, not available; PD, Parkinson disease. Magnetic Resonance Imaging No. of CGG Repeats Mild 69 corticosubcortical atrophy NA 61 Moderate, 20% None None Moderate subcortical and cerebellar atrophy 80 merase chain reaction technique containing the osmolyte, betaine, and primers c and f, as described by Saluto et al, 18 with only minor modifications to optimize betaine concentration (~2.2 M). Amplified DNA was then visualized on 2% agarose gels and ethidium bromide stained. Gray zone and premutation alleles were accurately sized on acrylamide gels. Hybridization was performed with a digoxigenin end-labeled oligonucleotide probe, (CGG) 10. Repeat sizes were determined using an Alpha Innotech FluorChem 8800 Image Detection System (Alpha Innotech Corp, San Leandro, California). Details of the method are presented in an article by Tassone et al. 19 MAIN OUTCOME MEASURE The sole outcome measure of this study is the number of CGG repeats, determined from the size of the polymerase chain reaction product. CGG repeats are considered to be premutation expansions if the size of the expansion exceeds 54 CGG repeats but is less than or equal to 200 CGG repeats. RESULTS Table 2. Screens for Premutation FMR1 Alleles Within Cohorts of Patients With Parkinson Disease or Parkinsonism Source (Center) Annesi 22 (Istituto Dermopatico dell Immacolata, Rome, Italy) Toft 23 (Mayo Clinic, Jacksonville, Florida) Deng 24 (Baylor College, Houston, Texas) Tan 25 (Singapore General Hospital, Singapore) Hedrich 26 (University of Lubeck, Lubeck, Germany) Current study (University of California, Davis) Abbreviation: NA, not applicable. Premutation Allele Frequency Male Female No. of CGG Repeats 0/203 NA NA 0/414 NA NA 0/216 NA NA 0/79 0/42 NA 1/265 1/108 Males, 55 Females, 63 3/902 NA 61, 69, 80 We screened 903 male patients with a diagnosis of primary parkinsonism or essential tremor for the presence of premutation expansions ( CGG repeats) of the FMR1 gene. Three individuals, all with the diagnosis of PD, were identified as carriers of premutation alleles of 61 (case 2), 69 (case 1), and 80 CGG repeats (case 3). The frequency of premutation alleles in this cohort was therefore 0.33% (3 of 903 patients), a value that is not significantly higher than values reported for the general population 12,20,21 and is consistent with previous, smaller screens of parkinsonism cohorts (Table 2). In addition, 18 gray-zone alleles (45-54 CGG repeats) were identified. Again, they were not significantly higher than values for the general population. The remaining 882 males harbored a normal allele ( 45 CGG repeats). The 3 male carriers of the FMR1 premutation did not show any unusual clinical features that would distinguish them from (noncarrier) patients with idiopathic PD. None of the 3 cases revealed features resembling FXTAS. In particular, they did not show cerebellar findings such as action tremor or gait ataxia, dysautonomia, or peripheral neuropathy. Relevant cognitive deficits were not present, though mild frontal lobe related deficits were reported, which is typical in advanced PD. Magnetic resonance images, available for 2 of 3 premutation carriers, did not demonstrate the FXTAS MCP finding, 6,27 though 1 individual did display mild cerebellar cortical atrophy. In addition, the premutation carriers did not have a family history positive for neurological or psychiatric disease, cognitive deficits, or development delays. Thus, it is likely that these cases represent coincidental findings. This suggestion is reinforced both by the relatively early ages at onset (34, 43, and 48 years; mean, 41.7 years) for the 3 FMR1 premutation carriers, which are much earlier than the mean age at onset for symptoms of FXTAS (62.6 years [SD, 8.1 years] for tremor; 63.6 years [SD, 7.3 years] for ataxia). 28 On the other hand, the early age at onset, which is generally associated with a stronger genetic predisposition, is an interesting finding and suggests that the FMR1 premutation may still have a role in the genetic susceptibility to common idiopathic PD. 1004

4 COMMENT In a screen of 903 cases with a prior diagnosis within the category of parkinsonism or essential tremor, there was no increase in the number of premutation FMR1 alleles over that which is expected in the general population, an observation that is not surprising in view of the differences in diagnostic features between patients with idiopathic PD and related disorders and patients with FXTAS. However, our study does underscore that testing for expanded FMR1 alleles within the parkinsonism movement disorder population is not justified in the absence of additional findings, such as cerebellar gait ataxia, MRI findings positive for MCP signs, or a family history of cognitive impairment, developmental delay, autism, or premature ovarian failure, which are all indicators of greater potential for expanded FMR1 alleles. There is one important caveat with the current findings, as with nearly all of the high-risk screens performed to date, namely, the selection bias intrinsic to the movement disorders population itself. In a retrospective study of known patients with FXTAS, only approximately 4% of patients were initially seen and had their syndrome diagnosed by a movement disorders specialist, 11 and so only a small fraction of the FXTAS cases would be expected to be found within cohorts of the type screened here and those presented in Table 2. Of the remaining 96% of cases identified by Hall et al, 11 approximately 70% were seen by general neurologists, and another 26% were seen by adult primary care physicians. Thus, there is a need for broader screens of older adult populations, both within and beyond the general neurology clinic. CONCLUSIONS A screen for expanded CGG-repeat alleles of the FMR1 gene within a cohort of 903 males with clinical presentations, including parkinsonism, detected 3 carriers of premutation alleles (61, 69, and 80 CGG repeats), a frequency (3 of 903 [0.33%]) that is not significantly higher than the frequency of premutation alleles in the general population (0.13%-0.39%). 20,21 The outcome of our study, the largest screen to date of individuals with parkinsonism, suggests that broad screening for premutation alleles in PD populations is unlikely to be productive in the absence of additional clinical or family history data suggestive of involvement of the FMR1 gene. Accepted for Publication: January 8, Correspondence: Flora Tassone, PhD, Department of Biochemistry and Molecular Medicine, University of California, Davis, School of Medicine, One Shields Avenue, Davis, CA (ftassone@ucdavis.edu). Author Contributions: Study concept and design: Kraff, Goldwurm, Hagerman, and Tassone. Acquisition of data: Kraff, Tang, Cilia, Canesi, Pezzoli, Goldwurm, and Tassone. Analysis and interpretation of data: Kraff, Hagerman, and Tassone. Drafting of the manuscript: Kraff, Goldwurm, Hagerman, and Tassone. Critical revision of the manuscript for important intellectual content: Kraff, Tang, Cilia, Canesi, Pezzoli, Goldwurm, Hagerman, and Tassone. Statistical analysis: Tassone. Obtained funding: Pezzoli, Goldwurm, Hagerman, and Tassone. Administrative, technical, and material support: Kraff, Tang, Cilia, Canesi, Goldwurm, Hagerman, and Tassone. Study supervision: Kraff, Goldwurm, Hagerman, and Tassone. Financial Disclosure: None reported. Funding/Support: This study was supported by the National Fragile X Foundation. DNA samples were obtained from the Human Genetic Bank of Patients Affected by Parkinson Disease and Parkinsonisms of the Parkinson Institute, Istituti Clinici di Perfezionamento, which is supported by grant GTF04007 from the Italian Telethon Foundation. Additional Contributions: We thank the patients and their relatives for their participation in this research. REFERENCES 1. Jacquemont S, Hagerman RJ, Leehey MA, et al. Penetrance of the fragile X associated tremor/ataxia syndrome in a premutation carrier population. JAMA. 2004; 291(4): Jacquemont S, Farzin F, Hall D, et al. Aging in individuals with the FMR1 mutation. Am J Ment Retard. 2004;109(2): Hagerman PJ, Hagerman RJ. Fragile X-associated tremor/ataxia syndrome (FXTAS). Ment Retard Dev Disabil Res Rev. 2004;10(1): Bacalman S, Farzin F, Bourgeois JA, et al. Psychiatric phenotype of the fragile X-associated tremor/ataxia syndrome (FXTAS) in males: newly described frontosubcortical dementia. J Clin Psychiatry. 2006;67(1): Grigsby J, Brega AG, Jacquemont S, et al. Impairment in the cognitive functioning of men with fragile X-associated tremor/ataxia syndrome (FXTAS). J Neurol Sci. 2006;248(1-2): Brunberg JA, Jacquemont S, Hagerman RJ, et al. Fragile X premutation carriers: characteristic MR imaging findings of adult male patients with progressive cerebellar and cognitive dysfunction. AJNR Am J Neuroradiol. 2002;23(10): Cohen S, Masyn K, Adams J, et al. Molecular and imaging correlates of the fragile X-associated tremor/ataxia syndrome. Neurology. 2006;67(8): Greco CM, Hagerman RJ, Tassone F, et al. Neuronal intranuclear inclusions in a new cerebellar tremor/ataxia syndrome among fragile X carriers. Brain. 2002; 125(pt 8): Greco CM, Berman RF, Martin RM, et al. Neuropathology of fragile X-associated tremor/ataxia syndrome (FXTAS). Brain. 2006;129(pt 1): Hagerman PJ, Hagerman RJ. The fragile-x premutation: a maturing perspective. Am J Hum Genet. 2004;74(5): Hall DA, Berry-Kravis E, Jacquemont S, et al. Initial diagnoses given to persons with the fragile X associated tremor/ataxia syndrome (FXTAS). Neurology. 2005; 65(2): Jacquemont S, Leehey MA, Hagerman RJ, Beckett LA, Hagerman PJ. Size bias of fragile X premutation alleles in late-onset movement disorders. J Med Genet. 2006;43(10): Hughes AJ, Daniel SE, Kilford L, Lees AJ. Accuracy of clinical diagnosis of idiopathic Parkinson s disease: a clinico-pathological study of 100 cases. J Neurol Neurosurg Psychiatry. 1992;55(3): Hughes AJ, Daniel SE, Lees AJ. Improved accuracy of clinical diagnosis of Lewy body Parkinson s disease. Neurology. 2001;57(8): Litvan I, Bhatia KP, Burn DJ, et al. Movement Disorders Society Scientific Issues Committee report: SIC Task Force appraisal of clinical diagnostic criteria for Parkinsonian disorders. Mov Disord. 2003;18(5): Elble RJ. Diagnostic criteria for essential tremor and differential diagnosis. Neurology. 2000;54(11)(suppl 4):S2-S Goldwurm S, Zini M, Di Fonzo A, et al. LRRK2 G2019S mutation and Parkinson s disease: a clinical, neuropsychological and neuropsychiatric study in a large Italian sample. Parkinsonism Relat Disord. 2006;12(7): Saluto A, Brussino A, Tassone F, et al. An enhanced polymerase chain reaction assay to detect pre- and full mutation alleles of the fragile X mental retardation 1 gene. J Mol Diagn. 2005;7(5): Tassone F, Hagerman RJ, Garcia-Arocena D, Khandjian EW, Greco CM, Hagerman PJ. Intranuclear inclusions in neural cells with premutation alleles in fragile X associated tremor/ataxia syndrome. J Med Genet. 2004;41(4):e

5 20. Dawson AJ, Chodirker BN, Chudley AE. Frequency of FMR1 premutations in a consecutive newborn population by PCR screening of Guthrie blood spots. Biochem Mol Med. 1995;56(1): Dombrowski C, Levesque S, Morel ML, Rouillard P, Morgan K, Rousseau F. Premutation and intermediate-size FMR1 alleles in males from the general population: loss of an AGG interruption is a late event in the generation of fragile X syndrome alleles. Hum Mol Genet. 2002;11(4): Annesi G, Nicoletti G, Tarantino P, et al. FRAXE intermediate alleles are associated with Parkinson s disease. Neurosci Lett. 2004;368(1): Toft M, Aasly J, Bisceglio G, et al. Parkinsonism, FXTAS, and FMR1 premutations. Mov Disord. 2005;20(2): Deng H, Le W, Jankovic J. Premutation alleles associated with Parkinson disease and essential tremor. JAMA. 2004;292(14): Tan EK, Zhao Y, Puong KY, et al. Expanded FMR1 alleles are rare in idiopathic Parkinson s disease. Neurogenetics. 2005;6(1): Hedrich K, Pramstaller PP, Stubke K, et al. Premutations in the FMR1 gene as a modifying factor in Parkin-associated Parkinson s disease? Mov Disord. 2005; 20(8): Jacquemont S, Hagerman RJ, Leehey M, et al. Fragile X premutation tremor/ ataxia syndrome: molecular, clinical, and neuroimaging correlates. Am J Hum Genet. 2003;72(4): Tassone F, Adams J, Berry-Kravis EM, et al. CGG correlates with age of onset of motor signs of the fragile X-associated tremor/ataxia syndrome (FXTAS) [published online ahead of print April 10, 2007]. Am J Med Genet B Neuropsychiatr Genet. 2007;144(4): Allingham-Hawkins DJ, Babul-Hirji R, Chitayat D, et al. Fragile X premutation is a significant risk factor for premature ovarian failure: the International Collaborative POF in Fragile X study, preliminary data. Am J Med Genet. 1999;83(4): Marozzi A, Vegetti W, Manfredini E, et al. Association between idiopathic premature ovarian failure and fragile X premutation. Hum Reprod. 2000;15(1): Sullivan AK, Marcus M, Epstein MP, et al. Association of FMR1 repeat size with ovarian dysfunction. Hum Reprod. 2005;20(2): Call for Papers Archives Express The Archives launched a new ARCHIVES Express section in the September 2000 issue. This section will enable the editors to publish highly selected papers within approximately 2 months of acceptance. We will consider only the most significant research, the top 1% of accepted papers, on new important insights into the pathogenesis of disease, brain function, and therapy. We encourage authors to send their most exceptional clinical or basic research, designating in the cover letter a request for expedited Archives Express review. We look forward to publishing your important new research in this accelerated manner. Roger N. Rosenberg, MD 1006