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Early Onset Parkinson's disease due to DJ1 mutations: An Indian study
Accepted Manuscript Early Onset Parkinson's disease due to DJ1 mutations: An Indian study Masoom M. Abbas, Shyla T. Govindappa, Sumedha Sudhaman, B.K. Thelma, Ramesh C. Juyal, Madhuri Behari, Uday B. Muthane PII:
S1353-8020(16)30128-6
DOI:
10.1016/j.parkreldis.2016.04.024
Reference:
PRD 2998
To appear in:
Parkinsonism and Related Disorders
Received Date: 9 December 2015 Revised Date:
6 April 2016
Accepted Date: 22 April 2016
Please cite this article as: Abbas MM, Govindappa ST, Sudhaman S, Thelma BK, Juyal RC, Behari M, Muthane UB, Early Onset Parkinson's disease due to DJ1 mutations: An Indian study, Parkinsonism and Related Disorders (2016), doi: 10.1016/j.parkreldis.2016.04.024. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Early Onset Parkinson’s Disease due to DJ1 Mutations: An Indian Study
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Authors: Dr. Masoom M Abbas1, DM Dr. Shyla T Govindappa1, MBBS Ms SumedhaSudhaman2, MSc Dr. B.K. Thelma2, PhD
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Dr Ramesh C Juyal3,PhD Dr. Madhuri Behari4#,DM
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Dr. Uday B Muthane1*, DM
1Parkinson’s and Ageing Research Foundation, No 58, C/12,Microlab Compound, Kudlu Village, Hosur Main Road, Bangalore 560068, India
2 Department of Genetics, University of Delhi, South Campus, Benito Juarez Road, New Delhi 110021, India
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3 Regional Centre for Biotechnology, Faridabad, Haryana, India
4 Professor &Head, Department of Neurology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi - 110029
# Present Affiliation-Department of Neurology, Fortis Flt Lt Rajan Dhall Hospital, Sector
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B, Pocket 1, Aruna Asaf Ali Marg, Vasant Kunj, New Delhi 110070, India *Corresponding Author- Medical Director, Parkinson’s and Ageing Research Foundation, No 58, C/12,Microlab Compound, Kudlu Village, Hosur Main Road,
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Bangalore 560068, India.
Tel:080-25744462. email: [email protected], [email protected] “Video is part of ms”
Word count -2067 Funding Agencies-Department of Biotechnology, Govt of India, New Delhi (#BT/PR2425/Med/13/089/2001,#BT/PR14500/MED/12/478/2010)
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ABSTRACT Introduction: Early Onset Parkinson’s Disease (EOPD) is genetically heterogeneous. PARK2 mutations are the commonest cause of autosomal recessive EOPD followed
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by PINK1.DJ1 mutations is rare and there is scarce literature on its phenotype and
long term outcome. Objectives: We undertook a retrospective study to determine the prevalence of DJ1 mutation(s) in an Indian population and describe the clinical features and long term outcome of EOPD patients with these mutations. Methods:
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One hundred EOPD patients and 114 controls were evaluated. All the seven
coding exons of DJ1 gene were screened for novel and reported mutations by
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PCR- Sanger sequencing. Results: A novel homozygous missense mutation (c.313 A>T, p. Ile105Phe) in exon 5 was seen in one patient and four unrelated patients had a homozygous missense single nucleotide variant rs71653619 (c.293 G>A, p.Arg98Gln). The clinical phenotype comprised of asymmetrical onset, slowly progressive Parkinsonism with levodopa induced
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motor restlessness in a patient with the novel mutation (c.313 A>T, p. Ile105Phe) while subjects with c.293 G>A, p.Arg98Gln had early onset levodopa responsive symmetrical Parkinsonism.Conclusion:DJ1 mutations account for ~5% of EOPD
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patients from the Indian population. This study further adds to the clinical spectrum of EOPD with DJ1 mutations.
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Key words: Early onset Parkinson’s disease, DJ1mutation, Phenomenology, Genetics
INTRODUCTION
Parkinson’s disease (PD) is multifactorial with both genetic and environmental factors playing a role. The genetic component is more important in patients with early age at onset. Early Onset Parkinson’s Disease or EOPD is defined as onset of parkinsonian features before 40 years and accounts for 3-5% of all PD cases [1]. It is classified into juvenile (before 21 years) and Young onset PD (21-40
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years) [1]. Autosomal recessive disorders producing EOPD are due to mutations in PARK 2, PINK1 (PARK 6) and DJ1 (PARK7) gene [2].ATP13A2 (PARK9), PLA2G6 (PARK14), FBXO7 (PARK15), SPG11 and POLG mutations are
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associated with atypical juvenile Parkinsonism [2]. DNAJC6 (PARK19) [3] and SYNJ1 (PARK20)[4] gene mutations are recent additions in autosomal recessive atypical juvenile PD.
In patients with early onset autosomal recessive Parkinson’s disease, PARK2
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mutations are most common accounting for around 77% of familial [5] and around 10-20% of EOPD patients in general [6].DJ1mutations are rare accounting for 1-
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2% of the sporadic cases with EOPD [7]. It was initially described in two consanguineous Dutch and Italian families [8].The gene is localized to chromosome 1p36 and comprises of seven coding exons encoding a DJ1 protein of 189 aminoacids [7].The DJ1protein is expressed in both cerebral and extra cerebral tissues and plays a role as a cellular sensor of oxidative stress [7].
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There is scant literature about the clinical phenotype and long term outcome in patients with DJ1 mutations because of its rarity [2, 7].About ten point mutations and deletions are described so far in homozygous and compound heterozygous
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state [7]. These mutations disrupt the normal protein structure thereby predisposing to proteosomal degradation and reducing their antioxidant activity
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and neuroprotective function [7]. Several studies have failed to identify DJ1 mutation(s) in large cohorts [9, 10]. There have been two Indian studies in this regard with one study showing a prevalence of
DJ1 variants to be 3.9% [11], while another study failed to identify any pathogenic mutations[12]. We undertook a retrospective analysis of EOPD patients with DJ1 mutations and followed them to assess their long term outcome.
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SUBJECTS AND METHODS Subjects: The study comprised of 100 EOPD patients (24 Juvenile PD and 76 EOPD) and 114 healthy controls of the same ethnicity. There were 15 familial
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cases (at least two affected members) in the cohort. These subjects were recruited from All India Institute of Medical Sciences (AIIMS), New Delhi and National Institute of Mental Health and Neurosciences (NIMHANS), Bangalore. The
institutional ethical committee of the two participating clinical centers approved
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the study and all participants signed an informed written consent form before enrolling in the study.
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PD was diagnosed using the UK Brain Bank Criteria [13] by a Movement Disorder specialist (UM in Bangalore and MB in New Delhi). PCR analysis was carried out to identify mutations in PARK2 [14], PINK1 (unpublished data), LRRK2 (G2019S, R1441C, R1441G and R1441H)[15],VPS35 and EIF4G1 [16] and were negative. Screening of familial PD patients for SNCA mutations was also negative [17].
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Mutation analysis: DNA was isolated from whole blood using standard DNA isolation technique. We screened all seven exons of DJ1 for novel and reported mutations by PCR- Sanger sequencing using a commercial sequencing facility.
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Seven exons of the gene along with exon-intron boundaries were amplified using seven pairs of intronic primers under standard PCR conditions (supplementary
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table). To investigate for the frequency of rs71653619 (c.293 G>A, p. Arg98Gln) variant we performed PCR followed by restriction fragment length polymorphism (RFLP) analysis in ethnically matched healthy controls. Further, the frequency of the same mutation was also checked in whole exome sequencing (WES) data from 218 control individuals, available in the laboratory. In patients with heterozygous mutations all seven coding sequences were screened for any additional mutations. Bioinformatics analysis: Mutations were screened after alignment of amplified
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DNA sequences against the reference sequences using the DNASTAR software package in the laboratory (DNASTAR, Madison,WI,USA).For multiple sequence alignments, protein sequences were aligned with paralogues and orthologues from
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the National Center for Biotechnology Information (NCBI) Structure Group conserved domain database [18]using ClustalW2 software. SIFT [19], PolyPhen2 [20] and mutation taster [21] softwares were used to investigate the pathogenicity of the mutations in sillico.
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RESULTS: Demographic Features
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The mean age of EOPD patients was 33+7years while it was 60+ 9years in the control group. There were 67% male patients in the study group compared to 50% in the controls. Genetic Analysis
A novel homozygous missense mutation c.313A>T, p. Ile105Phe in exon 5 was
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seen in a patient with sporadic EOPD. There were no other mutations in the remaining exons. This mutation was present in asymptomatic heterozygous state in his parents and his sister (figure 1). The remaining patients and 114 controls did
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not have this mutation. This mutation was predicted to be damaging by in silico tools such as SIFT [19], Poly Phen2 [20] and mutation taster [21].A brief clinical
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summary of this patient is presented in the following section. We also identified a homozygous variant rs71653619 (c.293 G>A, p. Arg98Gln {R98Q}) in exon 5in four unrelated EOPD patients. It was seen in heterozygous state in the unaffected parents of one of these four PD subjects (supplementary figure 1). This homozygous substitution was not seen in the entire control group but was observed in a heterozygous state in two control individuals based on their WES data. The demographic details of all the affected individuals are presented in table 1.
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Clinical Results PD patient with p.Ile105Phe mutation: This 24 year old gentleman presented with one and half years history of resting
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tremor in the right hand followed by left over next six months and generalized slowness in daily activities. He was born of second degree consanguineous
marriage and the family history was negative for parkinsonism. His younger sister died at the age of one year, the cause of which was not known (figure 1). On
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examination his cognition was normal, extraocular movements were full; speech was hypophonic and monotonous. Motor and sensory system examination was
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normal. He had a resting tremor (UPDRS grade 2) involving both upper limbs with generalized bradykinesia (Grade 1) and rigidity (Grade 1) in all four limbs. There was bilateral reduced arm swing and pull test was normal. Cerebellar signs were absent. His MRI Brain was normal. Autonomic function tests (tilt table test, heart rate and blood pressure variation to Valsalva maneuver, cold pressor, mental
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arithmetic, isometric contraction and deep breathing) were normal. He was managed with gradually increasing doses of ropinirole (12mg/day), trihexyphenidyl (22mg/day), amantadine (300mg/day) and clonazepam (2mg/day)
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over next five years with which he noticed only 5%improvement in his symptoms. Five years later the motor symptoms had progressed. His cognition was normal
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(MMSE 29/30) and depressive features (Beck Depression Inventory Score of 52/63) were noted. He had resting (Grade 3) and postural (Grade 3) tremor in bilateral upper limbs, with grade 2 bradykinesia and rigidity. His UPDRS motor score improved from 42 to 25 with levodopa/carbidopa (100/10mg). However he had extreme motor restlessness; he used to run uncontrollably, hit himself against the wall within half an hour after the dose and it lasted for around one hour (Supplementary video). It was disabling to such an extent that he had suicidal ideation. These symptoms recurred even with low dose of levodopa (half tablet of
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levodopa /carbidopa,100mg/ 10mg) because of which it had to be stopped. He was tried on Ayurvedic medications (Mucuna Pruriens and Ashwagandha) without much relief. Seven years later he required one person’s help for all his daily
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activities and succumbed to infection eleven years after the onset of symptoms. The clinical presentation of the four subjects with R98Q variant is summarized in table 2. DISCUSSION:
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In the present cohort we identified one patient with a novel homozygous
c.313A>T, p. Ile105Phe missense mutation in exon 5and four unrelated PD
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patients had rs71653619 (c.293 G>A, p. Arg98Gln) substitution which is reported to be a rare variant with possible functional relevance to PD pathology[22,23].The EOPD patient with novel p.Ile105Phe mutation presented with asymmetric onset, slowly progressive disease course of eleven years characterized by moderate levodopa response and levodopa sensitivity in form of motor restlessness. There
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were no pyramidal features or dystonia. The extreme motor restlessness even with low dose levodopa was a unique feature. Motor restlessness is labeled as a controversial entity in the clinical classification of levodopa induced dyskinesia. It
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is uncertain whether it represents levodopa induced dyskinesia or is a part of advanced disease [24]. One needs to distinguish it from akathisia which is a
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sensation of inner restlessness usually confined to lower limbs and without definite relation to ‘on’ or ‘off’ period [25]. The four EOPD patients with homozygous R98Q variant identified in this study (table 2) had asymmetrical PD symptoms with good levodopa response and levodopa induced dyskinesia. Psychiatric features like anxiety, depression and hallucinations were also noted. One patient had intermittent myoclonic jerks of bilateral upper limbs in addition to the tremor. This variant as mentioned in the results section was not found in 662 of the 664 individual chromosomes evaluated
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in the control population. Based on these observations, it is probable that R98Q is a pathogenic variant in the Indian population. Reports on the likely disruptive effect of this variant on the mitochondrial machinery [22], cellular oxidative stress
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response and consequential influence on disease onset even in the heterozygous state [23] lend support to our inference. However, in view of the extended
literature on the distribution of this variant in PD cases and controls discussion is warranted.
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R98Q variant is noted in pathologically proven late onset PD subjects with the clinical phenotype being similar to idiopathic PD [26].It is hypothesized that this
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variant is either non causal or acts as a susceptibility factor in a subset of late onset PD [26].On the other hand, in a few other studies, this rare variant is observed in control subjects as well as in PD cases [27, 28]. Considering PD is a late-onset disorder, it may be premature to dismiss the likely contribution of this
therein is available.
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variant to disease pathology until the current health status of controls screened
The phenotypic spectrum of patients with DJ1 mutations include early age at onset, slowly progressive asymmetrical levodopa responsive Parkinsonism with
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hyperreflexia, dystonia and psychiatric features [8]. Dementia and amyotrophic lateral sclerosis has been reported in an Italian family with a double homozygous
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mutation in exon 7 of theDJ1 gene [29].A novel mutation identified in our study had the phenotype as described previously in the literature [8]. Presence of motor restlessness with levodopa in this subject is noteworthy. This novel mutation needs to be tested in a larger EOPD patient cohort to further clarify its role in pathogenesis. DJ1 protein is widely expressed in brain and extra cerebral tissues. It was first identified as a novel oncogene in association with activated ras [30].It is involved in diverse cellular functions like oxidative phosphorylation, cellular
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transformation, RNA binding, androgen receptor signaling, spermatogenesis and fertilization[31].Experimental data suggests that DJ1protein has a stimulating effect on tyrosine hydroxylase and dopa decarboxylase enzymes involved in
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dopamine synthesis. The presence of mutations decreases this enzyme stimulating activity of DJ1protein [23]. This has been observed even with heterozygous
p.R98Q mutations suggesting a dominant negative effect [23]. Overexpression of +
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DJ1 protein enhances resistance against H2O2- and MPP induced cell damage while cells with p.R98Q, p.D149A and p.L166P mutations in the DJ1protein have
+
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impaired mitochondrial bioenergetics and increased vulnerability to H2O2- and MPP . Mitochondrial fragmentation and structural neuronal damage were also noted in these cells [22].
The prevalence of DJ1 mutations is low in EOPD patients compared to PARK2 and PINK1 globally[32]. However, in our population, though it is lower than PARK2
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mutation frequency[14],it is much higher (~5%)compared to Caucasian population (<1%).It is also comparable to another report from eastern India which found a novel p.Val35Ile homozygous DJ1mutation in two patients and heterozygous
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p.R98Q variant in six symptomatic patients [11].However, another study evaluating an independent PD cohort, from the same geographical region identified
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six intronic variants which are likely polymorphisms and no exonic variants in DJ1were noted [12].
In conclusion, contribution of DJ1 mutations to Autosomal Recessive EOPD seems to be modest in our population. It usually presents with slowly progressive dopamine responsive parkinsonian features associated with dystonia and psychiatric manifestations like depression, hallucinations and delusions. The presence of dopamine induced dyskinesia and sensitivity to levodopa as seen in our patient should alert the physician to the possible presence of DJ1 mutations
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especially in EOPD patients born of consanguineous parentage. References: 1. Schrag A, Schott JM. Epidemiological, clinical, and genetic
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characteristics of early-onset parkinsonism. Lancet Neurol 2006;5(4):355-363.
2. Vincenzo Bonifati. Autosomal recessive parkinsonism. Parkinsonism RelatDisord. 2012 Jan;18Suppl 1:S4-6.
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3. Edvardson S, Cinnamon Y, Ta-Shma A, Shaag A, Yim YI, Zenvirt S et al. A deleterious mutation in DNAJC6 encoding
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the neuronal-specific clathrin-uncoating co-chaperone auxilin, is associated with juvenile Parkinsonism. PLoS One2012; 7(5):e36458.
4. Quadri M, Fang M, Picillo M, Olgiati S, Breedveld GJ, Graafland J et al. Mutation in the SYNJ1 gene associated with autosomal recessive, early-
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onset parkinsonism. Human Mutation 2013; 34(9): 1208–1215. 5. Lucking CB, Durr A, Bonifati V, Vaughan J, De Michele G, Gasser T, Harhangi BS et al. Association between early-onset Parkinson’s disease and
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6. Klein C, Lohmann-Hedrich K. Impact of recent genetic findings in Parkinson’s disease. Curr Opin Neurol 2007;20: 453–464.
7. Christine Klein and Ana Westenberger. Genetics of Parkinson’s Disease. Cold Spring HarbPerspect Med 2012; 2(1): a008888.
8. van Duijn CM, Dekker MC, Bonifati V, Galjaard RJ, HouwingDuistermaat JJ, Snijders PJ et al. PARK7, a Novel Locus for Autosomal Recessive Early-Onset Parkinsonism on Chromosome 1p36. Am. J. Hum. Genet 2001;69:629–634
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9. Francesca Sironi, Paola Primignani, Sara Ricca, Sara Tunesi, Michela Zini, Silvana Tesei et al. DJ1 analysis in a large cohort of Italian early onset Parkinson Disease patients.
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Neurosci Lett 2013;12(17): 557(PB): 165–170. 10. Lockhart PJ, Bounds R, Hulihan M, Kachergus J, Lincoln S, Lin CH et al. Lack of mutations in DJ-1 in a cohort of Taiwanese ethnic Chinese with early-onset parkinsonism. MovDisord2004 ; 19(9):1065-1069.
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11. Sadhukhan T, Biswas A, Das SK, Ray K, Ray J.DJ-1 variants in Indian Parkinsons disease Patients. Disease Markers 2012;33: 127–135
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12. Sanyal J, Sarkar B, Banerjee TK, Mukherjee SC, Ray BC, Raghavendra Rao V. Evaluating intra-genetic variants of DJ-1 among Parkinson's disease patients of eastern India. Neurol Res 2011;33(4):349-53 13. Hughes AJ, Daniel SE, Kilford L, Lees AJ. Accuracy of clinical diagnosis of idiopathic Parkinson's disease: a clinico-pathological study of 100 cases.
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J Neurol Neurosurg Psychiatry 1992; 55(3):181-184. 14. Chaudhary S, Behari M, Dihana M, Swaminath PV, Govindappa ST, Jayaram S et al. Parkin mutations in familial and sporadic Parkinson's
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disease among Indians. Parkinsonism RelatDisord 2006;12(4):239-245. 15. Punia S, Behari M, Govindappa ST, Swaminath PV, Jayaram S, Goyal V
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et al. Absence/rarity of commonly reported LRRK2 mutations in Indian Parkinson’s disease patients. Neurosci Lett 2006;409(2):83–88 13. 15.
16. Sudhaman S, Behari M, Govindappa ST, Muthane UB, Juyal RC, Thelma BK. VPS35 and EIF4G1 mutations are rare in Parkinson's disease among
Indians. Neurobiol Aging 2013;34(10):2442. 17. Nagar S, Juyal RC, Chaudhary S, Behari M, Gupta M, Rao SN, Thelma BK. Mutations in the alpha-synuclein gene in Parkinson's disease among Indians. Acta Neurol Scand 2001;103(2):120-122
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18. NCBI conserved domain database. Available at: http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtm1 19. Kumar P, Henikoff S, Pauline C Ng . Predicting the effects of coding
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non-synonymous variants on protein function using the SIFT algorithm. Nat Protoc. 2009;4(7):1073-1081.
20. Ivan Adzhubei, Daniel M. Jordan,Shamil R.Sunyaev.
Predicting Functional Effect of Human Missense Mutations
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Using PolyPhen-2. CurrProtoc Hum Genet 2013; 76: 7.20. 21. Schwarz JM, Rödelsperger C, Schuelke M, Seelow D.
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Mutation Taster evaluates disease-causing potential of
sequence alterations. Nat Methods 2010;7(8):575-576. 22. Wang X, Petrie TG, Liu Y, Liu J, Fujioka H, Zhu X. Parkinson's diseaseassociated DJ-1 mutations impair mitochondrial dynamics and cause mitochondrial dysfunction. J Neurochem 2012;121(5):830-839.
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23. Ishikawa S, Taira T, Niki T, Takahashi-Niki K, Maita C, Maita H et al. Oxidative Status of DJ-1-dependent Activation of Dopamine Synthesis through Interaction of Tyrosine Hydroxylase and 4-Dihydroxy-L-
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phenylalanine (L-DOPA) Decarboxylase with DJ-1 J BiolChem 2009 16;284(42):28832-28844.
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24. Giovanni Fabbrini, Jonathan M. Brotchie, Francisco Grandas, Masahiro Nomoto, Christopher G. Goetz. Levodopa-Induced Dyskinesias. Movement Disorders 2007; 22:10:1379–1389. 25. Werner Poewe, Birgit Ho¨gl. Akathisia, restless legs and periodic limb movements in sleep in Parkinson’s disease. Neurology 2004;63:S12– S16. 26. Patrick M. Abou-Sleiman, Daniel G. Healy, Niall Quinn, Andrew J. Lees, Nicholas W. Wood. The Role of Pathogenic DJ-1 Mutations in
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Parkinson’s Disease. Ann Neurol 2003;54:283–286. 27. Hedrich K, Schäfer N, Hering R, Hagenah J, Lanthaler AJ, Schwinger E et al. The R98Q variation in DJ-1 represents a rare polymorphism. Ann
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Neurol 2004;55(1):145 28. Lockhart PJ, Lincoln S, Hulihan M, Kachergus J, Wilkes K, Bisceglio G, Mash DC, Farrer MJ. DJ-1 mutations are a rare cause of recessively
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inherited early onset parkinsonism mediated by loss of protein function. J
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Ragonese P et al. DJ-1 Mutations and Parkinsonism-DementiaAmyotrophic Lateral Sclerosis Complex. Ann Neurol 2005;58:803–807. 30. Nagakubo D, Taira T, Kitaura H, Ikeda M, Tamai K, Iguchi-Ariga SMM, and Ariga H. DJ-1, a novel oncogene which transforms mouse NIH3T3 cells in cooperation with ras. BiochemBiophys Res Commun 1987; 231: 509-513.
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32. Alcalay RN, Caccappolo E, Mejia-Santana H, Tang MX, Rosado L, Ross BM,et al. Frequency of known mutations in early onset PD;
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implication for genetic counseling: the CORE-PD study. Arch Neurol 2010; 67(9): 1116–1122.
Legends to video: Off period-Patient have features of Parkinson’s Disease with resting and postural tremor and bradykinesia. On period- Motor restlessness with uncontrollable motor movements are seen. Legends to figure 1: Pedigree chart of patient with p.Ile105Phe mutation. Squares
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represent men and circles represent women. Black square represent affected patient and black dots within represent the mutation in heterozygous state. Slashed symbol represents the deceased individuals.
with rs71653619 (c.293 G>A, p.Arg98Gln) variant
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Legends to Supplementary Figure: Sequencing profile of proband and parents
A- Sequencing profile of proband showing homozygous c.293 G>A, p.Arg98Gln; B, C- Sequencing profile of father and mother of the proband showing
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heterozygous c.293 G>A, p.Arg98Gln variant. Arrows indicate site of variation Acknowledgment
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We acknowledge Department of Biotechnology, Govt of India for financial assistance for the project and Parkinson's disease Society of Karnataka for providing free medications to the patient. We also acknowledge the late Ms Mitashree Das for PCR-based screening and analysis performed in this study
Authors' Roles
Dr. Uday B Muthane, Dr. MadhuriBehari, Prof. B.K. Thelma and Dr. Ramesh C.Juyal are
design,reviewand critique.
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involved in project conception, organization and execution, statistical and manuscript
Dr. Masoom M Abbas, Dr. Shyla T Govindappa and Ms Sumedha Sudhamanare involved in
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research project execution, statistical analysis design, execution, review and Critique, manuscript writing of the first draft, review and critique
Financial Disclosures
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Ms. Sumedha Sudhaman has received SRF from CSIR. Other authors do not have any financial
interests or potential conflicts of interest.
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Table 1: Demographic Features of Patients with DJ1 mutations Total(n)
Males
Females
Mean Age at Onset(Years)
Arg98Gln
4
3
1
28(+10.98)
Ile105Phe
1
1
0
22
Total
5
4
1
27(+9.85)
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Note: All the affected subjects were from South India
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Mutations
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Table 2: Phenotypic Features of patients with homozygous R98Q variant
Age at
Illness
Consang
Family
Clinic
Sy
Dopa
Dys
ID
Onset
Duration
uineous
history
al
mm
Response
kin
Featu
etri
(%)
esia
res
city
T,B,R
-
50
+
(years)
327076
14
15
+
-
LD 383295
35
23
-
+
T ,B,
-
367325
38
11
-
+
80
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R, Myo
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Patient
T, B,R
-
75
LD
*
326258
20
2
-
-
T,B,R
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LD
-
80
Remarks
Anxiety, RBD,
Depression, AFT-N +
Visual and Auditory Hallucinations
+
Blepharospasm, RBD, Depression Delusion, Visual Hallucinations, Depression
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Note: AFT-Autonomic Function Test, B- Bradykinesia, Myo-Myoclonus, LD- Limb Dystonia, R- Rigidity, RBD- REM Behavioral Disorder, T- Tremor, M- Male, F-Female. Family History: 383295- Father diagnosed to have PD at the age of 72 years. 367325 – Maternal uncle has PD at the age of 45 years. *- Lost for follow up after two years.
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HIGHLIGHTS
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• Early Onset Parkinson’s Disease is rare and usually due to parkin mutations • DJ1 mutations causing EOPD are rare • Presentation is with slowly progressive typical parkinsonian symptoms with good levodopa response. • A novel missense mutation p. Ile105Phe in exon 5 was noted in the present study • Extreme motor restlessness to levodopa was a unique feature in this patient with p. Ile105Phe mutation.
S1353-8020(16)30128-6
DOI:
10.1016/j.parkreldis.2016.04.024
Reference:
PRD 2998
To appear in:
Parkinsonism and Related Disorders
Received Date: 9 December 2015 Revised Date:
6 April 2016
Accepted Date: 22 April 2016
Please cite this article as: Abbas MM, Govindappa ST, Sudhaman S, Thelma BK, Juyal RC, Behari M, Muthane UB, Early Onset Parkinson's disease due to DJ1 mutations: An Indian study, Parkinsonism and Related Disorders (2016), doi: 10.1016/j.parkreldis.2016.04.024. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Early Onset Parkinson’s Disease due to DJ1 Mutations: An Indian Study
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Authors: Dr. Masoom M Abbas1, DM Dr. Shyla T Govindappa1, MBBS Ms SumedhaSudhaman2, MSc Dr. B.K. Thelma2, PhD
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Dr Ramesh C Juyal3,PhD Dr. Madhuri Behari4#,DM
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Dr. Uday B Muthane1*, DM
1Parkinson’s and Ageing Research Foundation, No 58, C/12,Microlab Compound, Kudlu Village, Hosur Main Road, Bangalore 560068, India
2 Department of Genetics, University of Delhi, South Campus, Benito Juarez Road, New Delhi 110021, India
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3 Regional Centre for Biotechnology, Faridabad, Haryana, India
4 Professor &Head, Department of Neurology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi - 110029
# Present Affiliation-Department of Neurology, Fortis Flt Lt Rajan Dhall Hospital, Sector
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B, Pocket 1, Aruna Asaf Ali Marg, Vasant Kunj, New Delhi 110070, India *Corresponding Author- Medical Director, Parkinson’s and Ageing Research Foundation, No 58, C/12,Microlab Compound, Kudlu Village, Hosur Main Road,
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Bangalore 560068, India.
Tel:080-25744462. email: [email protected], [email protected] “Video is part of ms”
Word count -2067 Funding Agencies-Department of Biotechnology, Govt of India, New Delhi (#BT/PR2425/Med/13/089/2001,#BT/PR14500/MED/12/478/2010)
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ABSTRACT Introduction: Early Onset Parkinson’s Disease (EOPD) is genetically heterogeneous. PARK2 mutations are the commonest cause of autosomal recessive EOPD followed
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by PINK1.DJ1 mutations is rare and there is scarce literature on its phenotype and
long term outcome. Objectives: We undertook a retrospective study to determine the prevalence of DJ1 mutation(s) in an Indian population and describe the clinical features and long term outcome of EOPD patients with these mutations. Methods:
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One hundred EOPD patients and 114 controls were evaluated. All the seven
coding exons of DJ1 gene were screened for novel and reported mutations by
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PCR- Sanger sequencing. Results: A novel homozygous missense mutation (c.313 A>T, p. Ile105Phe) in exon 5 was seen in one patient and four unrelated patients had a homozygous missense single nucleotide variant rs71653619 (c.293 G>A, p.Arg98Gln). The clinical phenotype comprised of asymmetrical onset, slowly progressive Parkinsonism with levodopa induced
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motor restlessness in a patient with the novel mutation (c.313 A>T, p. Ile105Phe) while subjects with c.293 G>A, p.Arg98Gln had early onset levodopa responsive symmetrical Parkinsonism.Conclusion:DJ1 mutations account for ~5% of EOPD
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patients from the Indian population. This study further adds to the clinical spectrum of EOPD with DJ1 mutations.
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Key words: Early onset Parkinson’s disease, DJ1mutation, Phenomenology, Genetics
INTRODUCTION
Parkinson’s disease (PD) is multifactorial with both genetic and environmental factors playing a role. The genetic component is more important in patients with early age at onset. Early Onset Parkinson’s Disease or EOPD is defined as onset of parkinsonian features before 40 years and accounts for 3-5% of all PD cases [1]. It is classified into juvenile (before 21 years) and Young onset PD (21-40
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years) [1]. Autosomal recessive disorders producing EOPD are due to mutations in PARK 2, PINK1 (PARK 6) and DJ1 (PARK7) gene [2].ATP13A2 (PARK9), PLA2G6 (PARK14), FBXO7 (PARK15), SPG11 and POLG mutations are
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associated with atypical juvenile Parkinsonism [2]. DNAJC6 (PARK19) [3] and SYNJ1 (PARK20)[4] gene mutations are recent additions in autosomal recessive atypical juvenile PD.
In patients with early onset autosomal recessive Parkinson’s disease, PARK2
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mutations are most common accounting for around 77% of familial [5] and around 10-20% of EOPD patients in general [6].DJ1mutations are rare accounting for 1-
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2% of the sporadic cases with EOPD [7]. It was initially described in two consanguineous Dutch and Italian families [8].The gene is localized to chromosome 1p36 and comprises of seven coding exons encoding a DJ1 protein of 189 aminoacids [7].The DJ1protein is expressed in both cerebral and extra cerebral tissues and plays a role as a cellular sensor of oxidative stress [7].
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There is scant literature about the clinical phenotype and long term outcome in patients with DJ1 mutations because of its rarity [2, 7].About ten point mutations and deletions are described so far in homozygous and compound heterozygous
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state [7]. These mutations disrupt the normal protein structure thereby predisposing to proteosomal degradation and reducing their antioxidant activity
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and neuroprotective function [7]. Several studies have failed to identify DJ1 mutation(s) in large cohorts [9, 10]. There have been two Indian studies in this regard with one study showing a prevalence of
DJ1 variants to be 3.9% [11], while another study failed to identify any pathogenic mutations[12]. We undertook a retrospective analysis of EOPD patients with DJ1 mutations and followed them to assess their long term outcome.
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SUBJECTS AND METHODS Subjects: The study comprised of 100 EOPD patients (24 Juvenile PD and 76 EOPD) and 114 healthy controls of the same ethnicity. There were 15 familial
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cases (at least two affected members) in the cohort. These subjects were recruited from All India Institute of Medical Sciences (AIIMS), New Delhi and National Institute of Mental Health and Neurosciences (NIMHANS), Bangalore. The
institutional ethical committee of the two participating clinical centers approved
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the study and all participants signed an informed written consent form before enrolling in the study.
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PD was diagnosed using the UK Brain Bank Criteria [13] by a Movement Disorder specialist (UM in Bangalore and MB in New Delhi). PCR analysis was carried out to identify mutations in PARK2 [14], PINK1 (unpublished data), LRRK2 (G2019S, R1441C, R1441G and R1441H)[15],VPS35 and EIF4G1 [16] and were negative. Screening of familial PD patients for SNCA mutations was also negative [17].
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Mutation analysis: DNA was isolated from whole blood using standard DNA isolation technique. We screened all seven exons of DJ1 for novel and reported mutations by PCR- Sanger sequencing using a commercial sequencing facility.
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Seven exons of the gene along with exon-intron boundaries were amplified using seven pairs of intronic primers under standard PCR conditions (supplementary
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table). To investigate for the frequency of rs71653619 (c.293 G>A, p. Arg98Gln) variant we performed PCR followed by restriction fragment length polymorphism (RFLP) analysis in ethnically matched healthy controls. Further, the frequency of the same mutation was also checked in whole exome sequencing (WES) data from 218 control individuals, available in the laboratory. In patients with heterozygous mutations all seven coding sequences were screened for any additional mutations. Bioinformatics analysis: Mutations were screened after alignment of amplified
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DNA sequences against the reference sequences using the DNASTAR software package in the laboratory (DNASTAR, Madison,WI,USA).For multiple sequence alignments, protein sequences were aligned with paralogues and orthologues from
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the National Center for Biotechnology Information (NCBI) Structure Group conserved domain database [18]using ClustalW2 software. SIFT [19], PolyPhen2 [20] and mutation taster [21] softwares were used to investigate the pathogenicity of the mutations in sillico.
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RESULTS: Demographic Features
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The mean age of EOPD patients was 33+7years while it was 60+ 9years in the control group. There were 67% male patients in the study group compared to 50% in the controls. Genetic Analysis
A novel homozygous missense mutation c.313A>T, p. Ile105Phe in exon 5 was
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seen in a patient with sporadic EOPD. There were no other mutations in the remaining exons. This mutation was present in asymptomatic heterozygous state in his parents and his sister (figure 1). The remaining patients and 114 controls did
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not have this mutation. This mutation was predicted to be damaging by in silico tools such as SIFT [19], Poly Phen2 [20] and mutation taster [21].A brief clinical
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summary of this patient is presented in the following section. We also identified a homozygous variant rs71653619 (c.293 G>A, p. Arg98Gln {R98Q}) in exon 5in four unrelated EOPD patients. It was seen in heterozygous state in the unaffected parents of one of these four PD subjects (supplementary figure 1). This homozygous substitution was not seen in the entire control group but was observed in a heterozygous state in two control individuals based on their WES data. The demographic details of all the affected individuals are presented in table 1.
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Clinical Results PD patient with p.Ile105Phe mutation: This 24 year old gentleman presented with one and half years history of resting
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tremor in the right hand followed by left over next six months and generalized slowness in daily activities. He was born of second degree consanguineous
marriage and the family history was negative for parkinsonism. His younger sister died at the age of one year, the cause of which was not known (figure 1). On
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examination his cognition was normal, extraocular movements were full; speech was hypophonic and monotonous. Motor and sensory system examination was
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normal. He had a resting tremor (UPDRS grade 2) involving both upper limbs with generalized bradykinesia (Grade 1) and rigidity (Grade 1) in all four limbs. There was bilateral reduced arm swing and pull test was normal. Cerebellar signs were absent. His MRI Brain was normal. Autonomic function tests (tilt table test, heart rate and blood pressure variation to Valsalva maneuver, cold pressor, mental
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arithmetic, isometric contraction and deep breathing) were normal. He was managed with gradually increasing doses of ropinirole (12mg/day), trihexyphenidyl (22mg/day), amantadine (300mg/day) and clonazepam (2mg/day)
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over next five years with which he noticed only 5%improvement in his symptoms. Five years later the motor symptoms had progressed. His cognition was normal
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(MMSE 29/30) and depressive features (Beck Depression Inventory Score of 52/63) were noted. He had resting (Grade 3) and postural (Grade 3) tremor in bilateral upper limbs, with grade 2 bradykinesia and rigidity. His UPDRS motor score improved from 42 to 25 with levodopa/carbidopa (100/10mg). However he had extreme motor restlessness; he used to run uncontrollably, hit himself against the wall within half an hour after the dose and it lasted for around one hour (Supplementary video). It was disabling to such an extent that he had suicidal ideation. These symptoms recurred even with low dose of levodopa (half tablet of
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levodopa /carbidopa,100mg/ 10mg) because of which it had to be stopped. He was tried on Ayurvedic medications (Mucuna Pruriens and Ashwagandha) without much relief. Seven years later he required one person’s help for all his daily
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activities and succumbed to infection eleven years after the onset of symptoms. The clinical presentation of the four subjects with R98Q variant is summarized in table 2. DISCUSSION:
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In the present cohort we identified one patient with a novel homozygous
c.313A>T, p. Ile105Phe missense mutation in exon 5and four unrelated PD
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patients had rs71653619 (c.293 G>A, p. Arg98Gln) substitution which is reported to be a rare variant with possible functional relevance to PD pathology[22,23].The EOPD patient with novel p.Ile105Phe mutation presented with asymmetric onset, slowly progressive disease course of eleven years characterized by moderate levodopa response and levodopa sensitivity in form of motor restlessness. There
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were no pyramidal features or dystonia. The extreme motor restlessness even with low dose levodopa was a unique feature. Motor restlessness is labeled as a controversial entity in the clinical classification of levodopa induced dyskinesia. It
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is uncertain whether it represents levodopa induced dyskinesia or is a part of advanced disease [24]. One needs to distinguish it from akathisia which is a
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sensation of inner restlessness usually confined to lower limbs and without definite relation to ‘on’ or ‘off’ period [25]. The four EOPD patients with homozygous R98Q variant identified in this study (table 2) had asymmetrical PD symptoms with good levodopa response and levodopa induced dyskinesia. Psychiatric features like anxiety, depression and hallucinations were also noted. One patient had intermittent myoclonic jerks of bilateral upper limbs in addition to the tremor. This variant as mentioned in the results section was not found in 662 of the 664 individual chromosomes evaluated
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in the control population. Based on these observations, it is probable that R98Q is a pathogenic variant in the Indian population. Reports on the likely disruptive effect of this variant on the mitochondrial machinery [22], cellular oxidative stress
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response and consequential influence on disease onset even in the heterozygous state [23] lend support to our inference. However, in view of the extended
literature on the distribution of this variant in PD cases and controls discussion is warranted.
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R98Q variant is noted in pathologically proven late onset PD subjects with the clinical phenotype being similar to idiopathic PD [26].It is hypothesized that this
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variant is either non causal or acts as a susceptibility factor in a subset of late onset PD [26].On the other hand, in a few other studies, this rare variant is observed in control subjects as well as in PD cases [27, 28]. Considering PD is a late-onset disorder, it may be premature to dismiss the likely contribution of this
therein is available.
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variant to disease pathology until the current health status of controls screened
The phenotypic spectrum of patients with DJ1 mutations include early age at onset, slowly progressive asymmetrical levodopa responsive Parkinsonism with
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hyperreflexia, dystonia and psychiatric features [8]. Dementia and amyotrophic lateral sclerosis has been reported in an Italian family with a double homozygous
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mutation in exon 7 of theDJ1 gene [29].A novel mutation identified in our study had the phenotype as described previously in the literature [8]. Presence of motor restlessness with levodopa in this subject is noteworthy. This novel mutation needs to be tested in a larger EOPD patient cohort to further clarify its role in pathogenesis. DJ1 protein is widely expressed in brain and extra cerebral tissues. It was first identified as a novel oncogene in association with activated ras [30].It is involved in diverse cellular functions like oxidative phosphorylation, cellular
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transformation, RNA binding, androgen receptor signaling, spermatogenesis and fertilization[31].Experimental data suggests that DJ1protein has a stimulating effect on tyrosine hydroxylase and dopa decarboxylase enzymes involved in
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dopamine synthesis. The presence of mutations decreases this enzyme stimulating activity of DJ1protein [23]. This has been observed even with heterozygous
p.R98Q mutations suggesting a dominant negative effect [23]. Overexpression of +
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DJ1 protein enhances resistance against H2O2- and MPP induced cell damage while cells with p.R98Q, p.D149A and p.L166P mutations in the DJ1protein have
+
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impaired mitochondrial bioenergetics and increased vulnerability to H2O2- and MPP . Mitochondrial fragmentation and structural neuronal damage were also noted in these cells [22].
The prevalence of DJ1 mutations is low in EOPD patients compared to PARK2 and PINK1 globally[32]. However, in our population, though it is lower than PARK2
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mutation frequency[14],it is much higher (~5%)compared to Caucasian population (<1%).It is also comparable to another report from eastern India which found a novel p.Val35Ile homozygous DJ1mutation in two patients and heterozygous
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p.R98Q variant in six symptomatic patients [11].However, another study evaluating an independent PD cohort, from the same geographical region identified
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six intronic variants which are likely polymorphisms and no exonic variants in DJ1were noted [12].
In conclusion, contribution of DJ1 mutations to Autosomal Recessive EOPD seems to be modest in our population. It usually presents with slowly progressive dopamine responsive parkinsonian features associated with dystonia and psychiatric manifestations like depression, hallucinations and delusions. The presence of dopamine induced dyskinesia and sensitivity to levodopa as seen in our patient should alert the physician to the possible presence of DJ1 mutations
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et al. Absence/rarity of commonly reported LRRK2 mutations in Indian Parkinson’s disease patients. Neurosci Lett 2006;409(2):83–88 13. 15.
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23. Ishikawa S, Taira T, Niki T, Takahashi-Niki K, Maita C, Maita H et al. Oxidative Status of DJ-1-dependent Activation of Dopamine Synthesis through Interaction of Tyrosine Hydroxylase and 4-Dihydroxy-L-
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Parkinson’s Disease. Ann Neurol 2003;54:283–286. 27. Hedrich K, Schäfer N, Hering R, Hagenah J, Lanthaler AJ, Schwinger E et al. The R98Q variation in DJ-1 represents a rare polymorphism. Ann
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Legends to video: Off period-Patient have features of Parkinson’s Disease with resting and postural tremor and bradykinesia. On period- Motor restlessness with uncontrollable motor movements are seen. Legends to figure 1: Pedigree chart of patient with p.Ile105Phe mutation. Squares
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represent men and circles represent women. Black square represent affected patient and black dots within represent the mutation in heterozygous state. Slashed symbol represents the deceased individuals.
with rs71653619 (c.293 G>A, p.Arg98Gln) variant
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Legends to Supplementary Figure: Sequencing profile of proband and parents
A- Sequencing profile of proband showing homozygous c.293 G>A, p.Arg98Gln; B, C- Sequencing profile of father and mother of the proband showing
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heterozygous c.293 G>A, p.Arg98Gln variant. Arrows indicate site of variation Acknowledgment
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We acknowledge Department of Biotechnology, Govt of India for financial assistance for the project and Parkinson's disease Society of Karnataka for providing free medications to the patient. We also acknowledge the late Ms Mitashree Das for PCR-based screening and analysis performed in this study
Authors' Roles
Dr. Uday B Muthane, Dr. MadhuriBehari, Prof. B.K. Thelma and Dr. Ramesh C.Juyal are
design,reviewand critique.
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involved in project conception, organization and execution, statistical and manuscript
Dr. Masoom M Abbas, Dr. Shyla T Govindappa and Ms Sumedha Sudhamanare involved in
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research project execution, statistical analysis design, execution, review and Critique, manuscript writing of the first draft, review and critique
Financial Disclosures
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Ms. Sumedha Sudhaman has received SRF from CSIR. Other authors do not have any financial
interests or potential conflicts of interest.
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Table 1: Demographic Features of Patients with DJ1 mutations Total(n)
Males
Females
Mean Age at Onset(Years)
Arg98Gln
4
3
1
28(+10.98)
Ile105Phe
1
1
0
22
Total
5
4
1
27(+9.85)
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Note: All the affected subjects were from South India
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Mutations
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Table 2: Phenotypic Features of patients with homozygous R98Q variant
Age at
Illness
Consang
Family
Clinic
Sy
Dopa
Dys
ID
Onset
Duration
uineous
history
al
mm
Response
kin
Featu
etri
(%)
esia
res
city
T,B,R
-
50
+
(years)
327076
14
15
+
-
LD 383295
35
23
-
+
T ,B,
-
367325
38
11
-
+
80
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R, Myo
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Patient
T, B,R
-
75
LD
*
326258
20
2
-
-
T,B,R
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LD
-
80
Remarks
Anxiety, RBD,
Depression, AFT-N +
Visual and Auditory Hallucinations
+
Blepharospasm, RBD, Depression Delusion, Visual Hallucinations, Depression
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Note: AFT-Autonomic Function Test, B- Bradykinesia, Myo-Myoclonus, LD- Limb Dystonia, R- Rigidity, RBD- REM Behavioral Disorder, T- Tremor, M- Male, F-Female. Family History: 383295- Father diagnosed to have PD at the age of 72 years. 367325 – Maternal uncle has PD at the age of 45 years. *- Lost for follow up after two years.
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HIGHLIGHTS
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• Early Onset Parkinson’s Disease is rare and usually due to parkin mutations • DJ1 mutations causing EOPD are rare • Presentation is with slowly progressive typical parkinsonian symptoms with good levodopa response. • A novel missense mutation p. Ile105Phe in exon 5 was noted in the present study • Extreme motor restlessness to levodopa was a unique feature in this patient with p. Ile105Phe mutation.