degeneration due to C9ORF72 hexanucleotide repeatexpansion: late onset psychotic clinical presentation.Biol Psychiatr 2013;74:384–91.

8 Snowden JS, Harris J, Richardson A, et al.Frontotemporal dementia with amyotrophic lateralsclerosis: a clinical comparison of patients with andwithout repeat expansions in C9ORF72. AmyotrophLateral Scler Frontotemporal Degener 2013;14:172–6.

9 Snowden JS, Rollinson S, Lafon C, et al. Psychosis,C9ORF72 and Dementia with Lewy bodies. J NeurolNeurosurg Psychiatry 2012;83:1031–2.

10 Claasen DO, Parisi JE, Giannini C, et al.Frontotemporal dementia mimicking dementia withLewy bodies. Cogn Behav Neurol 2008;21:157–63.

11 Cooper-Knock J, Frolov A, Highley JR, et al.C9ORF72 expansions, parkinsonism, and Parkinson’sdisease. Neurology 2013;81:808–11.

Parkin western blotting isuseful for identification ofpatients with Parkin-relatedParkinson’s disease

Parkinson’s disease (PD) is a progressiveneurological movement disorder charac-terised pathologically by degeneration ofdopaminergic neurons in the substantianigra pars compacta and the presence ofLewy bodies. Cardinal clinical features ofPD are bradykinesia, rigidity, restingtremor, postural instability and responsive-ness to levodopa. Identification of patientswith causative variations in the PD-related

genes (eg, SNCA, LRRK2, Parkin, PINK1,DJ-1 and ATP13A2) has been challenginggiven that only some patients have distinct-ive clinical phenotypes or a suggestivefamily history. Although clinical clues to aspecific monogenic form of PD may beobserved in some cases, for instance,behavioural problems and postural hypo-tension in patients with missense muta-tions in SNCA, early-onset dystonia inpatients with mutations in Parkin and verti-cal gaze palsy, spasticity and facial-faucalmini-myoclonus in patients with mutationsin ATP13A2,1 2 these manifestations arenot universally present. Moreover, patientswith mutations in LRRK2 or multiplicationsin SNCA can be clinically indistinguishablefrom idiopathic PD. Additionally, variabledisease expressivity3 and overlapping clin-ical presentations in the monogenic andidiopathic cases also complicate the diag-nostic process to identify each monogenicform of PD. Identification of mutations inParkin (MIM# 602544), which are thecommonest genetic cause of autosomalrecessive PD (MIM# 600116), requires acombination of direct sequencing andMLPA genetic analysis, given that missensemutations, microdeletions/duplications andlarge-scale rearrangements and gene dosageeffects can all cause Parkin-related PD.4

In this study, we determined Parkinexpression in PD patient-derived skin

fibroblasts, instead of Parkin-deficientblood leuckocytes, to investigate its usabil-ity in the diagnosis of Parkin-related PD.Western blotting of Parkin was performedin fibroblasts from individuals withknown mutations in PD-related genes (ie,Parkin, PINK1, ATP13A2, and LRRK2),idiopathic PD and healthy controls(figure 1A–C; detailed description of thesamples and method has previously beenpublished2 3 5).

Full-length wild-type Parkin protein(50 kDa) was present in all controls.Consistent with previous findings,6 7 theabsence of Parkin was observed in thehomozygous and compound heterozygousParkin mutations carriers. The expressionof Parkin was significantly decreased in thesingle heterozygous Parkin mutation car-riers to 58±4.5% compared to controls.Parkin was present in PINK1, ATP13A2,LRRK2 mutation carriers and in idiopathicPD patients at comparable levels to thehealthy controls (figure 1D). These resultsindicate that Parkin western blotting can beused to efficiently identify Parkin-relatedPD from other forms of monogenic PDthat may similarly present with an auto-somal recessive family history, younger ageof onset or overlapping phenotype. Thepossibility of patients harbouring missensemutations in Parkin cannot be excludedentirely by this method since Parkin levels

Figure 1 Determination of Parkin protein in human skin fibroblasts. Western blotting using whole-cell lysates from healthy controls, individualswith mutations in Parkinson’s disease (PD)-related genes and idiopathic PD. (A) Full-length wild-type Parkin protein (∼50 kDa) was absent inhomozygous and compound heterozygous Parkin mutation carriers. A reduced Parkin expression (∼50%) was observed in the single heterozygote,compared to the healthy controls. The expression of Parkin was comparable between the healthy controls and (B) PINK1, ATP13A2, (C) LRRK2mutation carriers and idiopathic PD; β-actin (42 kDa) was used as loading control. (D) Densitometric analysis of the immunoblots. The intensity ofeach band was normalised to β-actin and presented as mean±SD*; p<0.001 in two-tailed Student’s t test compared with the healthy controls.

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may not always be altered in this setting.8

Additionally, it can be used to confirm thefunctional deficit on next-generationsequencing protocols when a Parkin muta-tion has been identified.

In summary, western blotting, to iden-tify Parkin protein expression in skinfibroblast cultures, may be used as a sup-portive tool to identify patients suspectedof having mutations in the Parkin geneand justify further mutational analysis andgene dosage studies for the identificationof mutations in Parkin versus other genesthat cause monogenic young onsetParkinsonism. Further validation to fullyassess the usability of Parkin western blot-ting in differential diagnosis for Parkin-related PD using a larger cohort ofhealthy controls, idiopathic and mono-genic PD patients including with muta-tions in other PD-related genes (eg, SNCAand DJ-1) is warranted.

Brianada Koentjoro,1 Jin-Sung Park,1

Carolyn M Sue1,2

1Department of Neurogenetics, Kolling Institute ofMedical Research, Royal North Shore Hospital and theUniversity of Sydney, St. Leonards, New South Wales,Australia2Department of Neurology, Royal North Shore Hospital,St. Leonards, New South Wales, Australia

Correspondence to Professor Carolyn M Sue,Department of Neurology, Royal North Shore Hospitaland Kolling Institute of Medical Research, Universityof Sydney, St Leonards, NSW 2065, Australia;carolyn.sue@sydney.edu.au

Acknowledgements The authors thank the patientsfor participating in this project.

Contributors BK: drafting/revising the manuscript,study concept or design, analysis or interpretation ofdata, acquisition of data. Dr J-SP: drafting/revising themanuscript, study concept or design, analysis orinterpretation of data, study supervision orcoordination. Dr CMS: drafting/revising the manuscript,study concept or design, analysis or interpretation ofdata, study supervision or coordination, obtainingfunding.

Funding This work was supported by grants from theAustralian Brain Foundation and the Parkinson’s NewSouth Wales Association.

Competing interests CMS has been awarded anNHMRC Clinical Practitioner Fellowship and issupported by the Ramsay Health Teaching andResearch Fund.

Ethics approval Northern Sydney & Central CoastHealth Human Research Ethics Committee.

Provenance and peer review Not commissioned;externally peer reviewed.

To cite Koentjoro B, Park J-S, Sue C M. J NeurolNeurosurg Psychiatry 2014;85:1436–1437.

Received 19 March 2014Accepted 24 March 2014Published Online First 9 May 2014

J Neurol Neurosurg Psychiatry 2014;85:1436–1437.doi:10.1136/jnnp-2014-308142

REFERENCES1 Klein C, Lohmann-Hedrich K. Impact of recent genetic

findings in Parkinson’s disease. Curr Opin Neurol2007;20:453–64.

2 Park JS, Mehta P, Cooper AA, et al. Pathogenic effectsof novel mutations in the P-type ATPase ATP13A2(PARK9) causing Kufor-Rakeb syndrome, a form ofearly-onset parkinsonism. Hum Mutat2011;32:956–64.

3 Koentjoro B, Park JS, Ha AD, et al. Phenotypicvariability of parkin mutations in single kindred. MovDisord 2012;27:1299–303.

4 Hedrich K, Eskelson C, Wilmot B, et al. Distribution,type, and origin of Parkin mutations: review and casestudies. Mov Disord 2004;19:1146–57.

5 Huang Y, Halliday GM, Vandebona H, et al.Prevalence and clinical features of common LRRK2mutations in Australians with Parkinson’s disease.Mov Disord 2007;22:982–9.

6 Rakovic A, Grunewald A, Seibler P, et al. Effect ofendogenous mutant and wild-type PINK1 on Parkin infibroblasts from Parkinson disease patients. Hum MolGenet 2010;19:3124–37.

7 Pacelli C, De Rasmo D, Signorile A, et al.Mitochondrial defect and PGC-1alpha dysfunction inparkin-associated familial Parkinson’s disease. BiochimBiophys Acta 2011;1812:1041–53.

8 Gu WJ, Corti O, Araujo F, et al. The C289G andC418R missense mutations cause rapid sequestrationof human Parkin into insoluble aggregates. NeurobiolDis 2003;14:357–64.

A familial ALS case carryinga novel p.G147C SOD1heterozygous missensemutation with non-executivecognitive impairment

Amyotrophic lateral sclerosis (ALS) is afatal motor neuron disease causing pro-gressive muscle weakness and wasting.Death usually occurs within 3–5 yearsfrom respiratory failure. Approximately10% of cases are familial (fALS).The fre-quency of SOD1 gene mutations inpatients with fALS varies among popula-tions, from 0% in Ireland to 13.6% inItaly and 23.5% in Scandinavia.1 SOD1encodes for the Cu/Zn SuperoxideDismutase 1. Mutations are usually auto-somal dominant, but the p.D90A and thep.D96N may be autosomal recessive.2

Performing the genetic screening of anItalian fALS series, we found a novelc.442g>t heterozygous missense mutationof SOD1 gene leading to a substitution ofcysteine for glycine (p.G147C). Suchmutation was absent in healthy controls(n=130). The patient provided writteninformed consent.The index case displayed progressive

weakness and wasting of both hands whenhe was 52 years old. One year after the

onset he also exhibited tongue hypotro-phy with fasciculations, spastic parapar-esis, impairment of feet extension andbrisk jaw jerk and lower limbs reflexes.Plantar response was absent bilaterally. Hereferred diffuse cramps and fasciculations.Dysphagia, dysarthria, dysphonia and dys-pnoea were absent. The amyotrophiclateral sclerosis funcional rating scalerevised (ALSFRS-R) was 44/48. Needleelectromyography (EMG) showed chronicand active denervation in bulbar andspinal regions. Forced vital capacity was106%. The neuropsychological evaluationshowed an impaired performance in theRey-Osterrieth Complex Figure (ROCF)Test, in copy and recall task, while othertests had normal scores. Brain MRIshowed selective atrophy of the rightsupramarginal gyrus (figure 1A), slighthyperintensity of the corticospinal tractsin T2-weighted scans, reduced fractionalanisotropy along the right corticospinaltract in diffusion tensor imaging (DTI)scans. Cervical cord MRI was normal.18F-FDG cerebral PET revealed reduceduptake (p=0.001) in the right supramar-ginal gyrus (Brodmann area (BA) 40)(figure 1B). Brain MRI and 18F-Fludeoxyglucose (18F-FDG) positronemission tomography (PET) and neuro-psychological assessment were repeated6 months later. The focal atrophy of theright supramarginal gyrus resultedunchanged at MRI. 18F-FDG PETshowed slight extension of the area ofhypometabolism previously observed atthis site and the appearance of a newarea in the right frontopolar region(p=0.01) (figure 1C). A relative reduc-tion of the uptake in the right caudatenucleus, probably due to deafferen-tation, seems to support the significativ-ity of the frontopolar hypometabolism.The neuropsychological evaluation con-firmed the deficit in the ROCF anddemonstrated a reduction of the scoresof mini mental state examination(MMSE), trail making test (TMT)-A andTMT-B, Clock Test and frontal assess-ment battery (FAB), although they werestill normal.

Two brothers of the proband died fromALS. The former showed muscle weaknessand wasting at upper limbs when he was46 years of age. He had a rapid worseningand died from respiratory failure10 months after the onset. The latterreported cramps at lower limbs when hewas 48 years of age, followed by weaknessof the left upper limb. He showed aclassic ALS phenotype and died fromrespiratory failure 27 months after theonset. Two siblings were 46 years and

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