ARTICLES ATP13A2 missense mutations in juvenile parkinsonism and young onset Parkinson disease

ARTICLES ATP13A2 missense mutations in juvenile parkinsonism and young onset Parkinson disease A. Di Fonzo, MD H.F. Chien, MD M. Socal, MD S. Giraudo, BS C. Tassorelli, MD G. Iliceto, MD G. Fabbrini, MD R. Marconi, MD E. Fincati, MD G. Abbruzzese, MD P. Marini, MD F. Squitieri, MD M.W. Horstink, MD, PhD P. Montagna, MD A. Dalla Libera, MD F. Stocchi, MD S. Goldwurm, MD, PhD J.J. Ferreira, MD G. Meco, MD E. Martignoni, MD L. Lopiano, MD L.B. Jardim, MD, PhD B.A. Oostra, PhD E.R. Barbosa, MD The Italian Parkinson Genetics Network* V. Bonifati, MD, PhD Address correspondence and reprint requests to Dr. V. Bonifati, Department of Clinical Genetics, Erasmus MC, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands v.bonifati@erasmusmc.nl See also page 1553 Supplemental data at www.neurology.org ABSTRACT Objective: To assess the prevalence, nature, and associated phenotypes of ATP13A2 gene mutations among patients with juvenile parkinsonism (onset 21 years) or young onset (between 21 and 40 years) Parkinson disease (YOPD). Methods: We studied 46 patients, mostly from Italy or Brazil, including 11 with juvenile parkinsonism and 35 with YOPD. Thirty-three cases were sporadic and 13 had positive family history compatible with autosomal recessive inheritance. Forty-two had only parkinsonian signs, while four (all juvenile-onset) had multisystemic involvement. The whole ATP13A2 coding region (29 exons) and exon-intron boundaries were sequenced from genomic DNA. Results: A novel homozygous missense mutation (Gly504Arg) was identified in one sporadic case from Brazil with juvenile parkinsonism. This patient had symptoms onset at age 12, levodopa-responsive severe akinetic-rigid parkinsonism, levodopa-induced motor fluctuations and dyskinesias, severe visual hallucinations, and supranuclear vertical gaze paresis, but no pyramidal deficit nor dementia. Brain CT scan showed moderate diffuse atrophy. Furthermore, two Italian cases with YOPD without atypical features carried a novel missense mutation (Thr12Met, Gly533Arg) in single heterozygous state. Conclusions: We confirm that ATP13A2 homozygous mutations are associated with human parkinsonism, and expand the associated genotypic and clinical spectrum, by describing a homozygous missense mutation in this gene in a patient with a phenotype milder than that initially associated with ATP13A2 mutations (Kufor-Rakeb syndrome). Our data also suggest that ATP13A2 single heterozygous mutations might be etiologically relevant for patients with YOPD and further studies of this gene in Parkinson disease are warranted. NEUROLOGY 2007;68:1557 1562 The term juvenile parkinsonism is conventionally used to indicate patients with onset of a parkinsonian syndrome before the age of 21 years. If onset is between age of 21 and 40 years, the term young onset Parkinson disease (YOPD) is preferred. 1,2 A distinct juvenile multisystemic neurodegenerative disease was described in 1994 in a consanguineous family from Jordan (Kufor-Rakeb disease, KRD). 3 Five siblings had akineticrigid parkinsonism of subacute, juvenile onset (around age 13) which responded to levodopa, pyramidal tract dysfunction, supranuclear gaze paresis, and dementia. Despite the presence of a good response to levodopa, this syndrome is therefore distinct from PD, and close (though not identical to) the rare Davison pallido-pyramidal degeneration. 4 A more recent clinical follow-up study of the KRD family described the presence of additional features such as visual hallucinations, facial-faucial-finger mini-myoclonus (FFF), and oculogyric dystonic *Members of the Italian Parkinson Genetics Network are listed in the appendix. From the Department of Clinical Genetics (A. Di Fonzo, B.A.O., V.B.), Erasmus MC, Rotterdam, The Netherlands; Department of Neurology (H.F.C., E.R.B.), University of São Paulo, Brazil; Hospital de Clínicas de Porto Alegre (M.S., L.B.J.), Brazil; Department of Neuroscience (S. Giraudo, L.L.), University of Turin, Italy; Institute IRCCS Mondino (C.T.), Pavia, Italy; Department of Neurology (G.I.), University of Bari, Italy; Department of Neurological Sciences (G.F., G.M.), La Sapienza University, Rome; Neurology Division (R.M.), Misericordia Hospital, Grosseto, Italy; Department of Neurology (E.F.), University of Verona, Italy; Department of Neurosciences (G.A.), Ophthalmology & Genetics, University of Genova, Italy; Department of Neurology (P. Marini), University of Florence, Italy; Neurogenetics Unit (F. Squitieri), IRCCS Neuromed, Pozzilli, Italy; Department of Neurology (M.W.H.), Radboud University Nijmegen Medical Center, Netherlands; Department of Neurology (P. Montagna), University of Bologna, Italy; Neurology Division (A. Dalla Libera), Boldrini Hospital, Thiene, Italy; IRCCS San Raffaele Pisana (F. Stocchi), Rome, Italy; Parkinson Institute (S. Goldwurm), Istituti Clinici di Perfezionamento, Milan; Neurological Clinical Research Unit (J.J.F.), Institute of Molecular Medicine, Lisbon, Portugal; A. Avogadro University (E.M.), Novara, and Institute IRCCS S. Maugeri, Scientific Institute of Veruno, Italy; and Department of Neurological Sciences, University of Milan, and Foundation Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena (A. Di Fonzo), Milan, Italy. The Italian control DNA samples were from the Human Genetic Bank of Patients Affected by PD and Parkinsonisms, Parkinson Institute Istituti Clinici di Perfezionamento, Milan, supported by the Italian Telethon Foundation (grant GTF04007). Supported by a research grant from the Internationaal Parkinson Fonds (The Netherlands) to V.B. Disclosure: The authors report no conflicts of interest. Copyright 2007 by AAN Enterprises, Inc. 1557

(A) Electropherograms of fragments of the ATP13A2 genomic sequence. The position of the mutations identified in this study is indicated. The corresponding sequence from unrelated healthy controls is provided as a reference. (B) Conservation of the ATP13A2 protein residues targeted by the mutations identified in patients. The closest homologues of the ATP13A2 protein were aligned using the program ClustalW. GenBank accession numbers are as follows: NP_071372.1 (ATPase type 13A2, Homo sapiens); XP_001087655.1 (similar to ATPase type 13A2 isoform 2, Macaca mulatta); NP_083373.1 (ATPase type 13A2, Mus musculus); XP_342963.3 (similar to ATPase type 13A2, Rattus norvegicus); AAH77611.1 (MGC84593 protein, Xenopus laevis); AAN06554.2 (Q8IMA, Drosophila melanogaster). (C) Schematic representation of the ATP13A2 protein, its predicted functional domains, and position of the mutations identified in this study. The homozygous mutation (Gly504Arg) is underlined. Figure 1 Molecular findings spasms. 5 The pathology of KRD remains unexplored, but neuroimaging showed progressive, diffuse brain atrophy. 3,5 A genetic locus was mapped to the chromosome 1p36 region in the original KRD family and termed PARK9. 6 Linkage was recently confirmed in a second pedigree originating from Chile with a similar phenotype, and pathogenic, homozygous, and compound heterozygous loss-of-function mutations in the ATP13A2 gene were identified in these two kindreds. 7 Whether ATP13A2 mutations are also associated with pure juvenile parkinsonism or YOPD remains currently unknown. We sequenced the complete ATP13A2 coding region in 46 patients with parkinsonism of juvenile or young onset, and we identified three novel ATP13A2 missense mutations, including one homozygous mutation. METHODS Forty-six index patients from two cohorts of consecutively collected familial and sporadic cases were included in this study. Thirteen patients were from families compatible with autosomal recessive inheritance of parkinsonism ( 2 affected siblings and unaffected parents) and onset before age 40 years (average: 29, range 10 to 40 years) (AR cohort). Four of these cases had onset before 21 years (juvenile parkinsonism) and the remaining nine had YOPD. Among these families, eight were from Italy, four from Brazil, and one from The Netherlands. The remaining 33 patients had sporadic parkinsonism (no first-degree relatives affected by PD) and disease onset before age 33 years (average 26, range 10 to 33 years). Seven of these cases had juvenile parkinsonism and 26 had YOPD. Among the sporadic cases, 22 were from Italy, 10 from Brazil, and 1 from Japan. Parental consanguinity was reported in three of the AR families and in three sporadic cases. Mutations in the parkin, 8 PINK1, 9 and DJ-1 10 gene were previously excluded in all the familial and most of the sporadic cases included in this study. Forty-two cases (35 with YOPD and 7 juvenile parkinsonism) had a pure parkinsonian syndrome (bradykinesia and at least one of the following: resting tremor, rigidity, and postural instability); progressive course; positive response to dopaminergic therapy; and no atypical features or known causes of parkinsonism. The remaining four index cases were two familial (one Italian and one Dutch) and two sporadic Brazilian patients with juvenile parkinsonism and additional clinical features such as pyramidal signs, supranuclear gaze paresis, or cognitive deterioration, resembling thereby the pallidopyramidal degeneration or KRD. Neurologic examination included the Unified Parkinson s Disease Rating Scale (UPDRS, motor part), 11 Hoehn-Yahr scale, and Mini-Mental State Examination. The novel sequence variants detected in patients were tested in ethnically matched healthy controls (140 Italian and 109 Portuguese subjects, mainly spouses of patients) free from PD or dementia, aged 50 years and recruited at the Milan and Lisbon centers. Moreover, 78 additional unrelated Brazilian patients with early-onset PD, recruited at São Paulo and Porto Alegre centers, were screened for the mutation found in one Brazilian index. The relevant ethical authorities approved the study and written informed consent was obtained from all subjects. Genomic DNA was isolated from peripheral blood using standard protocols. The 29 exons and intron-exon boundaries of the ATP13A2 gene were amplified using PCR. For sequencing of some exons, additional internal primers were also used. All primer sequences, PCR conditions, and sequencing methods are reported in table E-1 on the Neurology Web site at www. neurology.org. The mutations were numbered from the A of the ATG-translation initiation codon, and the consequences of mutations at the protein level were predicted according to the ATP13A2 mrna sequence (GenBank accession number NM_022089.1) and protein sequence (accession number NP_071372.1). RESULTS In one sporadic patient from Brazil (BR- 3042), genomic sequencing of ATP13A2 revealed a novel homozygous g1510c mutation in exon 15, predicted to cause the missense Gly504Arg change in the encoded protein (figure 1A). The presence of the mutation in heterozygous state was confirmed in both his unaffected parents. Furthermore, novel single heterozygous missense mutations were found in two Italian cases: c35t in exon 2, predicted protein effect: Thr12Met in one sporadic case (VE-29); and g1597a in exon 16, pre- 1558 Neurology 68 May 8, 2007

Table Clinical features in patients with ATP13A2 mutations Patient BR-3042 Patient VE-29 Patient PK-69-01 Mutation Gly504Arg Thr12Met Gly533Arg Zygosity Homozygous Heterozygous Heterozygous Gender Male Male Male Onset age, y 12 30 40 Duration, y 10 5 16 UPDRS 65 33 NA Bradykinesia Rigidity Tremor in extremities Postural instability NA Asymmetric onset L-Dopa response Motor fluctuations NA Dyskinesias NA Hallucinations Psychotic episodes Supranuclear gaze paresis Babinski sign Dementia Brain imaging Diffuse atrophy (CT) Normal (MRI) Normal (MRI) Other features Lip/chin tremor Sleep benefit Obsessive-compulsive disturbance UPDRS Unified Parkinson s Disease Rating Scale; NA not available. dicted protein effect: Gly533Arg, in one familial patient (PK-69-01) (figure 1A). In these cases, direct sequencing of genomic DNA did not detect additional variants. Due to the lack of mrna samples, cdna studies could not be performed. One brother of the PK-69-01 case was also affected by earlyonset PD, but DNA samples for co-segregation studies were not available. A novel exonic, silent variant (c1005t, predicted protein effect: Ala335Ala) was detected in heterozygous state in one patient (TOR-97). Many known single nucleotide polymorphisms (SNPs) were also detected in several cases in heterozygous or homozygous state, but none of these leads to amino acid change in the protein: c1815t (Pro605Pro), c2637t (Gly879Gly), g2790a (Ser930Ser), g2970a (Val990Val), c3192t (Ala1064Ala), and g3516a (Pro1172Pro). The Gly504Arg mutation was not observed in 498 chromosomes from healthy individuals, including 218 from the Portuguese and 280 from the Italian population. Moreover, this mutation was absent from 156 chromosomes from Brazilian patients with early-onset PD. The remaining two missense mutations were not found in 240 chromosomes from healthy Italian individuals. Furthermore, the missense mutations identified in this study all replace highly conserved residues in the ATP13A2 protein (figure 1B). The most important clinical features in the patients carrying ATP13A2 mutations are reported in the table. CASE REPORT The patient with homozygous missense ATP13A2 mutation (BR-3042) is the youngest of four siblings born from a nonconsanguineous Brazilian couple of white ethnicity. They were unable to trace back the exact geographic origin of their ancestors. No other cases of parkinsonism are reported in the family. He had normal developmental milestones, finished high school smoothly, and, even after many years of disease and severe motor disability, he applied for college and passed the examinations with excellent grades. At the age of 12 the subject s family, friends, and teachers noticed that he was very slow and his movements were awkward and clumsy. One year after the onset of symptoms, a diagnosis of juvenile PD was made. In 2002, 6 years later, he was seen by some of us (H.F.C., E.R.B.) at the Department of Neurology, University of São Paulo. He showed an initial very good response to levodopa and bromocriptine. However, soon thereafter he developed choreic dyskinesias and visual hallucinations, which Neurology 68 May 8, 2007 1559

Images obtained in the patient with homozygous ATP13A2 mutation, after 10 years of disease course. A diffuse moderate atrophy is evident in both the cerebral and cerebellar structures. Figure 2 Brain CT images worsened over months and were later accompanied by episodes of aggressive behavior, for which he required hospitalization and treatment with haloperidol. Bromocriptine was then stopped and a combination of only levodopa/benserazide and atypical antipsychotics (quetiapine) allowed satisfactory control of motor and psychic symptoms. Over the last year the parkinsonism worsened with re-emergence of hallucinations and severe fluctuations and dyskinesias, requiring repeated changes in the drug schedule, including the combination of entacapone and levodopa. The therapeutic window is now narrowed to the extent that he has alternating periods of severe akinetic-rigid states (when off ) and severe dyskinesias and psychiatric disturbances (when on ). He never had tremor in his extremities. Currently, his mental status remains very good: he scored 29 on MMSE and was very well informed on political facts. However, detailed neuropsychological testing could not be performed. To prevent side effects, he is currently taking only small amounts of drugs (levodopa [plus benserazide] 350 mg/day; quetiapine 50 mg/day), and his neurologic examination is dominated by severe akinetic-rigid parkinsonism (H-Y stage V; UPDRS motor score 65/108). A limitation in upward gaze is also present. Deep tendon reflexes are brisk but Babinski sign is absent. He has no sphincter problems. There is no tremor in the extremities. A very mild tremor is noticed in the lower lip and chin when he is talking, but other movement disorders such as the FFF described in patients with KRD 5 are not observed. A brain CT scan performed in 2006, after 10 years of disease course, showed diffuse moderate atrophy in both the cerebral and cerebellar structures (figure 2). DISCUSSION We provide independent confirmation that homozygous ATP13A2 gene mutations are associated with human neurodegeneration. We also expand the genotypic spectrum describing missense mutations in this gene. In contrast, the mutations reported in the two original families with KRD were more dramatic, leading to premature truncation or in-frame absence of a long fragment of the encoded protein. 7 The Gly504Arg is not an obvious loss-offunction mutation, and the absence of other PD cases in the family precluded further co-segregation study. Due to this, it is difficult to conclusively prove pathogenicity. However, several arguments strongly support the view that this mutation is disease causing in the BR-3042 patient. We did not observe the Gly504Arg mutation, even in heterozygous state, in any of the controls or any other Brazilian patients with PD (total: 654 chromosomes tested), while it is present in homozygous state in the BR- 3042 patient and in heterozygous state in both his unaffected parents. The Gly504 residue is highly conserved in the known mammalian homologues of the ATP13A2 protein (figure 1B), and is located in the larger cytosolic loop close to the predicted catalytic phosphorylation site (figure 1C). The mutation introduces a non-conservative change, by replacing glycine, a small neutral amino acid, with the large and positively charged arginine. This modification might therefore affect the biochemical properties of this region. The interpretation of the role of the two mutations found in single heterozygous state in the two YOPD cases is not simple. The Thr12Met and Gly533Arg mutations are both non-conservative amino acid substitutions, targeting highly conserved residues (figure 1B) and as such, have potential pathogenic relevance. The replacement of threonine12 with methionine might also introduce a novel translation initiation codon, leading to the skipping of the first 11 residues in the ATP13A2 protein. Furthermore, these mutations were not identified in control chromosomes, indicating that they are not common polymorphisms. Interestingly, despite the large size of the ATP13A2 protein, we observed not a single frequent missense polymorphism, while all of the frequent exonic SNPs detected were silent variants. This suggests that the ATP13A2 protein has low polymorphism content, 1560 Neurology 68 May 8, 2007

at least in the studied populations, adding further relevance to the missense mutations we identified. One possibility is that these mutations are disease-causing in trans with a second mutation, which has escaped detection by the methods used in this study. We did not perform cdna or gene dosage analysis and therefore we could have missed large genomic rearrangements, or mutations in promoter or other regulatory regions. As another possibility, these mutations might act as risk factor for development of YOPD, in combination with mutations in other genes. The screening of the parkin, PINK1, and DJ-1 gene was negative in our three cases with ATP13A2 mutations. Finally, the ATP13A2 mutations might be unrelated to the disease in these two patients, as heterozygous carriers of recessive mutations exist in the population, and are expected to be detected if a sufficient number of individuals is tested. However, this last possibility is unlikely on the basis of the low frequency of juvenile parkinsonism and the even lower frequency of cases attributable to homozygous mutations in this gene. The same debate about the role of single heterozygous mutations applies to the other genes causing recessive forms of YOPD (parkin, PINK1, DJ-1). 9,12-14 Taken as a whole, our data suggest therefore that mutations in the ATP13A2 gene might be relevant for YOPD. Whether mutations or polymorphisms in the ATP13A2 gene are associated with later-onset PD remains unknown and this also deserves further study. ATP13A2 encodes a large, 1,180 amino acids transmembrane protein belonging to the Group 5 P-type ATPase class and displaying lysosomal localization in overexpression studies. 7 The function and substrate specificity of this protein remain unknown, but, intriguingly, the ATP13A2 mrna is highly expressed in the brain, particularly in substantia nigra, and it seems upregulated in the brain of patients with the common late-onset idiopathic PD. 7 Other research lines point to an important role for lysosomes in the pathogenesis of common, lateonset PD. The lysosomes are important for the degradation of the alpha-synuclein protein, 15,16 and heterozygous mutations in the GBA gene, encoding the lysosomal enzyme glucocerebrosidase, are emerging as an important risk factor for Lewy body disorders (PD and Lewy body dementia). 17,18 The clinical phenotype in the original families with homozygous ATP13A2 mutations (KRD) was characterized by additional multisystemic features, such as pyramidal tract signs, supranuclear gaze palsy, and severe cognitive decline. 3,5 On the contrary, in our homozygous case the atypical features were limited to an impaired upward gaze and to moderate brain atrophy. The early motor and psychiatric complications induced by levodopa narrow the therapeutic window, and brisk tendon reflexes are present in this patient; however, these are all common features in juvenile parkinsonism and YOPD, including those caused by parkin mutation. 19 The subacute onset of symptoms described in the original Jordan KRD family was not reported in our case or in the Chilean KRD family. Our case illustrates therefore that homozygous ATP13A2 gene mutations are not invariably associated with the full-blown KRD, but might be found in patients with a milder clinical presentation. One of the patients of the Chilean family described in the original cloning report (Case II-9) also displayed incomplete clinical presentation, with lack of Babinski sign and supranuclear gaze palsy, though severe cognitive deterioration was present in addition to parkinsonism. 7 A milder phenotype could correlate with presence of missense mutations and residual protein function, while the full-blown KRD could be more frequent in those carrying truncating mutations. However, a larger number of cases and mutations are needed to test this hypothesis. The identification of the missense mutation described in this study and of other patients with mutations in this gene might foster the understanding of the function of the ATP13A2 protein, and of the mechanisms of disease caused by its dysfunction. Screening of the ATP13A2 gene should be included in the diagnostic workup of juvenile parkinsonism, especially in, but not limited to, the cases with additional clinical features, such as pyramidal signs, cognitive deterioration, or supranuclear gaze paresis. ACKNOWLEDGMENT The authors thank all patients and relatives for their contributions, and Tom de Vries-Lentsch, Erasmus MC, Rotterdam, for artwork. APPENDIX Members of the Italian Parkinson Genetics Network are as follows: V. Bonifati, Erasmus MC, Rotterdam, The Netherlands; N. Vanacore, National Centre of Epidemiology, Rome; G. Meco, G. Fabbrini, E. Fabrizio, N. Locuratolo, C. Scoppetta, M. Manfredi, A. Berardelli, University La Sapienza, Rome; L. Lopiano, S. Giraudo, B. Bergamasco, University of Torino; C. Tassorelli, C. Pacchetti, G. Nappi, IRCCS Mondino, Pavia; S. Goldwurm, A. Antonini, G. Pezzoli, Parkinson Institute, Istituti Clinici di Perfezionamento, Milan; G. Riboldazzi, G. Bono, Insubria University, Varese; F. Raudino, Hospital Valduce, Como; Mi. Manfredi, Poliambulanza Hospital, Brescia; E. Fincati, University of Verona; M. Tinazzi, A. Bonizzato, Hospital Borgo Trento, Verona; C. Ferracci, Hospital of Belluno; A. Dalla Libera, Boldrini Hospital, Thiene; G. Abbruzzese, R. Marchese, University of Genova; P. Montagna, University of Bologna; P. Marini, F. Massaro, University of Firenze; R. Marconi, Misericordia Hospital, Grosseto; M. Guidi, INRCA Institute, Ancona; C. Minardi, F. Rasi, Bufalini Hospital, Cesena; M. Onofrj, A. Thomas, University of Chieti; F. Stocchi, L. Vacca, IRCCS San Raffaele Pisana, Rome; F. De Pandis, Villa Margherita Hospital, Benevento; M. De Mari, C. Diroma, G. Iliceto, P. Lamberti, University of Bari; V. Toni, G. Trianni, Hospital of Casarano; A. Mauro, Hospital of Salerno; A. De Gaetano, Hos- Neurology 68 May 8, 2007 1561

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