a novel mutation of the ε-sarcoglycan gene in a chinese family with myoclonus-dystonia syndrome

Brief Reports

Entacapone Improves Absorptionof a Coadministered Salt in

Patients with Parkinson’s Disease

Thomas Muller, MD,1,2* Dirk Woitalla, MD,2

Oliver Goetze, MD,3,4 and Christoph Erdmann, MD2

1Department of Neurology, St. Joseph Hospital Berlin-Weißensee, Berlin, Germany; 2Department of Neurology, St.Josef Hospital, Ruhr University Bochum, Bochum, Germany;

3Department of Medicine I, St. Josef Hospital, Ruhr-UniversityBochum, Bochum, Germany; 4University Hospital Zurich,Division of Gastroenterology and Hepatology, Zurich,

Switzerland

Abstract: Entacapone (EN) improves the efficacy of levo-dopa/dopadecarboxylase inhibitor (LD/DDI) formulationsby inhibition of the enzyme catechol-O-methyltransferase(COMT). COMT inhibition also promotes the synthesis ofbasic LD metabolites, whereas DDI support the composi-tion of acidic LD derivatives. LD metabolism correlates tothe one of 13C-sodium-octanoate, which is employed inbreath tests to measure gastric emptying velocity. Objec-tives were to investigate the impact of COMT inhibitionon the recovery rate of 13C-sodium-octanoate in parkinso-nian patients, who received first 100 mg LD/Carbidopa(CD) and the next day 100 mg LD/CD/EN combined with13C-sodium-octanoate in each case. The recovery rate of

13C-sodium-octanoate was significant higher during theLD/CD/EN–compared with the LD/CD condition. COMTinhibition combined with LD/DDI improves absorption ofa co-administered salt probably due to a COMT inhibi-tion induced basic environment in gastrointestinal mem-branes. This improves dissolution and absorption of acidsand salts. Thus it may enhance absorption of LD itself. �2008 Movement Disorder Society

Key words: entacapone; levodopa; absorption; Parkin-son’s disease

Patients with Parkinson’s disease (PD) often receive

a drug combination therapy that involves multiple daily

doses of a particular compound and is supplemented

with other drugs at least partially sharing the modes of

action. Efficacy of all these compounds depend on

patient compliance, the nature of the delivery system,

physicochemical properties of the drug and physiologi-

cal considerations. Each of them is interrelated to the

other ones and affects the rate at which the drug is

absorbed throughout the gastrointestinal tract and thus

its bioavailability and pharmacokinetic profile.1 In this

respect, Entacapone (EN) improves the properties and

dose of levodopa/dopadecarboxylase inhibitor (LD/

DDI) by inhibition of peripheral LD metabolism via

the enzyme catechol-O-methyltransferase (COMT).

However, COMT inhibition may also promote the syn-

thesis of more basic LD metabolites, i.e., the tyrosine

aminotransferase dependent substrates dihydroxypheny-

lpyruvate acetate and trihydroxyphenylacetate. There-

fore COMT inhibition may model the environmental

pH and the physicochemical properties of LD for its

duodenal absorption, since this enzyme is located in

higher concentrations in the membranes of the gastro-

intestinal tract.1–3 Generally, these physicochemical

properties of a drug also affect its absorption through

the gastrointestinal tract.2,4 Many compounds, includ-

ing LD, are weak bases or weak acids or are the salts

of them and as such demonstrate pH dependent solubil-

ity. The pH partition hypothesis asserts that the pas-

sage rate of a drug through a membrane is dependent

on the environmental pH and pKa of the drug. Drugs

with low pKa are nonionized in the stomach and sub-

sequently rapidly absorbed. On passage to the small

intestine with comparatively increased pH, the rate of

ionization is changed and absorption subsequently

slowed. The converse is true for drugs with higher pKa

value. This influences the bioavailability of hydrophilic

drug formulations, which have a narrow window of

absorption, limited predominantly to the stomach or

the upper intestine as limited by their low pKa value

and/or the site of active transport absorption mecha-

nism, like LD.1,2,5 The gastrointestinal absorption

behavior of orally administered LD also depends on

gastrointestinal transit rates, since absorption of LD

occurs mainly in the proximal third of the small intes-

tine (duodenum/jejunum) but not in the stomach.3,6

*Correspondence to: Dr. Thomas Muller, Department of Neurol-ogy, St. Joseph Hospital Berlin-Weißensee, Gartenstr. 1, 13088Berlin, Germany. E-mail: [email protected] [email protected]

Received 14 January 2008; Revised 8 April 2008; Accepted 12May 2008

Published online 10 June 2008 in Wiley InterScience (www.

interscience.wiley.com). DOI: 10.1002/mds.22176

1458

Movement DisordersVol. 23, No. 10, 2008, pp. 1458–1482� 2008 Movement Disorder Society

Intestinal LD absorption is rapid and complete, but the

plasma bioavailability of LD is only 30% due to prior

degradation to dopamine by dopa decarboxylase and

to a lesser extent to 3-O-methyldopa (3-OMD) by

COMT, i.e., in the gut membranes. The longer LD is

retained in the stomach and in the small intestine the

more extensively it is metabolized and made less avail-

able for absorption.6–8 A compound, which shares the

peripheral absorption site profile of LD, is sodium-

octanoate, which is employed as 13C marked substrate

in breath tests as a noninvasive feasible alternative

method without ionizing radiation to measure gastric

emptying of solids and liquids. After intake, 13C-

sodium-octanoate is rapidly absorbed from the proxi-

mal intestine and carried to the liver via the portal

venous system. There it is oxidized and eliminated as

CO2 in the breath, reflecting gastric emptying as the

rate limiting step of the process.9 Accordingly, signifi-

cant associations between pharmacokinetic plasma

behavior of LD and the outcomes of the 13C-octanoic

acid breath test (OBT) appeared. In this study no impact

of EN addition on gastric emptying time was found.6

Objectives of the present exploratory trial were to

investigate the impact of COMT inhibition on the re-

covery rate of 13C-sodium-octanoate in PD patients.

SUBJECTS AND METHODS

Subjects

Fourteen idiopathic PD patients [age: 57.46 6 9.31,

40–73 (mean 6 SD, range) years; 9 men, 5 women,

daily LD/DCI dosage: 282.14 6 216.27, 0–600 mg,

Hoehn and Yahr scale stage: 2.54 6 0.37, II–III; Uni-

fied Parkinson’s Disease Rating Scale (UPDRS) I: 4.14

6 1.29, 2–6; UPDRS II: 13.92 6 2.81, 9–19; UPDRS

III: 25.29 6 10.49, 12–48; UPDRS IV: 1.07 6 0.9, 0–

3)] without unpredictable fluctuations and without

additional disorders participated. No patient had a his-

tory of gastrointestinal disorders and had undergone

abdominal surgery (except appendectomy), or took

compounds, i.e., domperidone, which model gastroin-

testinal motility or acidity before or during the study.

Design

All participants performed the OBT in a standar-

dized setting at 7 a.m. on two consecutive days. On

day one LD/Carbidopa (LD/CD) (o.i.d., 100 mg

Nacom) and on day two LD/CD/EN (o.i.d., 100 mg

Stalevo) were administered together with 100 mL

water 30 minutes after intake of the solid OBT meal.

Application of additional antiparkinsonian drug treat-

ment remained stable on both investigation days after

the study.

Determination of 13C-Sodium-Octanoate Absorption

One egg was mixed with 13C-sodium-octanoate (100

mg, Euriso-top, Saint-Aubin Cedex, France; chemical

and isotopic purity >99.5%) to label the solid compo-

nent of the test meal. This was served as breakfast af-

ter an overnight fast as an egg omelet together with

60 g of white bread, 5 g of margarine, and 200 mL of

water (53% carbohydrate, 27% lipid, 20% protein;

250 kcal). Thirty minutes before baseline and then at

certain moments (see Fig. 1), breath specimens were

FIG. 1. Recovery of 13C-sodium-octanoate. Percentage 13C-sodium-octanoate dose recovered (PDR in %) after ingestion of a labeled solidmeal 30 minutes before oral administration of levodopa/carbidopa (LD/CD) or levodopa/carbidopa/entacapone (LD/CD/EN) at 0 minutes. SEM,standard error of mean.

1459ENTACAPONE IMPROVES ABSORPTION

Movement Disorders, Vol. 23, No. 10, 2008

sampled into gas-tight plastic bags. The participants

were kept in a relaxed sitting position and physical ac-

tivity was restricted during the tests. All subjects ate

their test meals within 10 minutes. The 13CO2/12CO2

isotopic ratio was determined within 24 hour using iso-

tope-selective infrared spectrometry (NDIRS; Wagner

Analysentechnik, Bremen, Germany). The results were

assessed as previously.6,9 To measure the proportion of

the substrate given by mouth that is metabolized the

results were expressed as maximal percentage dose of13C recovered (PDRmax) and percentage dose of 13C

recovered (PDR) over time for each time interval.

Evaluation of OBT parameters gastric emptying coeffi-

cient, gastric lag phase, gastric half emptying time and

time point of maximal 13CO2 exhalation were done

like in previous trials.6,9

Statistics

The comparison of the LD/CD with the LD/CD/EN

condition was performed with the t-test for dependent

samples.

Ethics

All participants gave written informed consent. The

study was approved by the local ethic committee of

the university.

RESULTS

The PDR values were significant (P < 0.0001)

higher during the LD/CD/EN condition (6.96 6 3.78)

compared with the LD/CD condition (6.13 6 4.28)

(see Fig. 1). Patterns of gastric emptying were similar

in both groups. There was no difference of the com-

puted gastric emptying parameters between the LD/CD

and the LD/CD/EN condition (Table 1). No side effects

were reported.

DISCUSSION

This study showed that COMT inhibition with

EN increases the recovery rate of the salt 13C-sodium-

octanoate. It is known, that medicaments are better

absorbed with a rise of the pH value due to the dra-

matically better increased solubility. This results in a

complete dissolution of the substance, that makes them

readily available for absorption.1,5 This resembles to

our study scenario. LD administered in the traditional

form only with a DDI is mainly metabolized to deriva-

tives which lower the pH value. The main substrate is

vanillic acid. This one is formed from the initial tyro-

sine aminotransferase dependent derivative dihydroxy-

phenylpyruvate followed by a COMT dependent sec-

ond metabolic step. An alternative pathway for the for-

mation of vanillic acid is the COMT triggered

degradation of LD to 3-OMD with a subsequent tyro-

sine aminotransferase dependent second step. A further

metabolite is homovanillic acid, since this one depends

on the initial DDI dependent conversion to dopamine.

However this LD derivative may play a minor role,

since we administered the DDI CD during both study

conditions. Dopamine is metabolized to the initial

MAO dependent dihydroxyphenylacetic acid or as an

alternative to the COMT dependent 3-methroxytyr-

amine. Both, dihydroxyphenylacetic acid via COMT

and 3-methroxytyramine via monoaminooxidases are

then transformed to homovanillic acid. During the LD/

CD/EN condition, LD is mainly degraded to the tyro-

sine aminotransferase dependent substrates dihydroxy-

phenylpyruvate acetate and trihydroxyphenylacetate

and to a distinct lesser extent to dihydroxyphenylacetic

acid in the periphery. This more basic milieu may

hypothetically influence the activity of the intestinal

H1-coupled nutrient, micronutrient and drug transport-

ers in the mammalian small intestine and thus enable a

better transport of acids into the peripheral blood circu-

lation.2,4,5

We confirm, that there is no effect of acute COMT

inhibition with EN on gastric emptying.6 However, our

present design does not exclude, that repeat application

of EN may influence the gastric emptying rate and fur-

ther increase the bioavailability of coadministered sub-

stances. In the case of LD, this may cause a deterio-

rated degradation and result in higher LD concentra-

tions in the gastrointestinal membranes due to the

simultaneous blocking of COMT by EN and DDI by

CD. Since LD itself may contribute to delayed gastric

emptying rate, this results in the more smooth increase

of LD concentrations after repeat EN application.6,7

Limitations of this trial are that we performed no

cross over design due to the short half life of a singular

EN administration, and that we did not try to measure

the gastrointestinal pH value in the membranes or sim-

ply in the gut itself due to ethical and technical reasons.

TABLE 1. Gastric emptying parameters

LD/CD LD/CD/EN P value

GEC 2.78 6 1.04 3.16 6 0.65 0.29tlag (min) 118.1 6 38.77 102.6 6 20.3 0.29t50 (min) 166.1 6 39.82 152.7 6 29.75 0.43tpeak (min) 110 6 39.61 92.14 6 26.65 0.25

Data are expressed as means 6 standard deviation. GEC, gastricemptying coefficient; tlag, gastric lag phase; t50, gastric half emptyingtime; tpeak, time point of maximal 13CO2 exhalation.

1460 T. MULLER ET AL.


In conclusion, COMT inhibition combined with LD/

DDI improves absorption of a coadministered salt

probably due to a COMT inhibition induced basic

environment in gastrointestinal membranes. This

improves dissolution and absorption of acids and salts.

Thus it may enhance absorption of LD itself.2,4,5

Acknowledgments: OG was supported by the Deutsche For-schungsgemeinschaft (Go 1358/1-1). We thank Tanja Steinerand Christine Stamm for technical assistance.

REFERENCES

1. Kortejarvi H, Urtti A, Yliperttula M. Pharmacokinetic simulation ofbiowaiver criteria: The effects of gastric emptying, dissolution,absorption and elimination rates. Eur J Pharma Sci 2007;30:155–166.

2. Forsberg MM, Huotari M, Savolainen J, Mannisto PT. The role ofphysicochemical properties of entacapone and tolcapone on theirefficacy during local intrastriatal administration. Eur J Pharma Sci2005;24:503–511.

3. Goetze O, Wieczorek J, Muller T, Przuntek H, Schmidt WE, Woi-talla D. Impaired gastric emptying of a solid test meal in patientswith Parkinson’s disease using 13C-sodium octanoate breath test.Neurosci Lett 2005;375:170–173.

4. Lennernas H. Modeling gastrointestinal drug absorption requiresmore in vivo biopharmaceutical data: experience from in vivo dis-solution and permeability studies in humans. Curr Drug Metab2007;8:1389–2002.

5. Thwaites DT, Anderson CMH. H1-coupled nutrient, micronutrientand drug transporters in the mammalian small intestine. ExpPhysiol 2007;92:603–619.

6. Muller T, Erdmann C, Bremen D, et al. Impact of gastric emptyingon levodopa pharmacokinetics in Parkinson disease patients. ClinNeuropharmacol 2006;29:61–67.

7. Muller T, Erdmann C, Muhlack S, et al. Pharmacokinetic behaviourof levodopa and 3-O-methyldopa after repeat administration of lev-odopa/carbidopa with and without entacapone in patients with Par-kinson’s disease. J Neural Transm 2006;113:1441�1448.

8. Woitalla D, Goetze O, Kim JI, et al. Levodopa availabilityimproves with progression of Parkinson’s disease. J Neurol2006;253:1221�1226.

9. Goetze O, Nikodem AB, Wiezcorek J, et al. Predictors of gastricemptying in Parkinson’s disease. Neurogastroenterol Motil 2006;18:369�375.

Familial Parkinsonism withDigenic Parkin and PINK1

Mutations

Manabu Funayama, PhD,1 Yuanzhe Li, MD,2

Tak-Hong Tsoi, FHKAM, FRCP,3

Ching-Wan Lam, MBChB, PhD,4

Takekazu Ohi, MD, PhD,5 Shogo Yazawa, MD, PhD,6

Eiichiro Uyama, MD, PhD,7 Ruth Djaldetti, MD,8

Eldad Melamed, MD,8 Hiroyo Yoshino, BS,1

Yoko Imamichi,2 Hiroshi Takashima, MD, PhD,9

Kenya Nishioka, MD, PhD,2

Kenichi Sato, MD, PhD,10 Hiroyuki Tomiyama, MD,2

Shin-Ichiro Kubo, MD, PhD,2

Yoshikuni Mizuno, MD,1 andNobutaka Hattori, MD, PhD1,2*

1Research Institute for Diseases of Old Age, JuntendoUniversity School of Medicine, Bunkyo-Ku, Tokyo, Japan;2Department of Neurology, Juntendo University School ofMedicine, Bunkyo-Ku, Tokyo, Japan; 3Department ofMedicine, Pamela Youde Nethersole Eastern Hospital,

Hong Kong, China; 4Department of Chemical Pathology, TheChinese University of Hong Kong, Prince of Wales

Hospital, Hong Kong, China; 5Department of Neurology,Kurashiki Central Hospital, Kurashiki, Okayama, Japan;6Department of Neurology, Miyazaki Prefectural Hospital of

Nobeoka, Nobeoka, Miyazaki, Japan; 7Department of Neurology,Graduate School of Medical Sciences, Kumamoto University,Kumamoto, Kumamoto, Japan; 8Department of Neurology,Rabin Medical Center, Beilinson Campus, Petah Tiqva,

Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv,Israel; 9Department of Neurology and Geriatrics,

Kagoshima University School of Medicine, Kagoshima,Japan; 10Department of Neurology, Juntendo University

Nerima Hospital, Nerima-Ku, Tokyo, Japan

Abstract: To clarify the genetic correlation between parkinand PINK1, we screened for PINK1 mutations in 175parkinsonism patients with parkin mutations. We detectedtwo sibling pairs and one sporadic patient carrying bothparkin and PINK1 mutations. The age at onset of Parkin-sonism of patients with the digenic mutations was lowerthan that of patients with the same parkin mutation alone.In addition, two of three patients carrying both parkinand PINK1 mutations had schizophrenia. These findingsindicate that PINK1 mutation might modify parkin muta-tion-positive Parkinsonism, and PINK1 mutations might

*Correspondence to: Dr. Nobutaka Hattori, Department of Neurol-ogy, Juntendo University Graduate School of Medicine and DentalSciences, Bunkyo-Ku, Tokyo 113-8421, Japan.E-mail: [email protected]

Received 1 November 2007; Revised 9 April 2008; Accepted 25April 2008



1461DIGENIC MUTATION OF PARKIN AND PINK1


be associated withpsychiatric disorders. � 2008 MovementDisorder Society

Key words: Parkinson’s disease; parkin; PINK1; digenic;psychiatric disorder

Parkinson’s disease (PD) is one of the most frequent

neurodegenerative disorders caused by loss of dopami-

nergic neurons in the substantia nigra, which results

in decreased dopamine availability in the striatum.

Although most cases with PD are sporadic, several

genes are associated with the monogenic forms of Par-

kinsonism and related disorders. Identification of the

causative genes and their functions in these rare forms

of the disease can provide tremendous insights into the

pathogenesis of PD and opens up new areas of medical

research on this disease.

Parkin [MIM 602544; PARK2] and PTEN-inducedputative kinase 1 (PINK1) [MIM 608309; PARK6]have been reported as the causative genes of PARK2-and PARK6-linked autosomal recessive parkinsonism

(ARP), respectively.1 Intriguingly, several lines of

evidence suggest that heterozygous mutations of parkinand PINK1 could play a role in the development of

parkinsonism despite the fact that they were originally

identified as the responsible genes for ARP.2,3 In addi-

tion, parkin and PINK1 mutations might be associated

with psychiatric disorders.1,4,5 Thus, these results sug-

gest the importance of these genes in sporadic PD as

well as psychiatric disorders, in addition to ARP.

Recent biochemical and morphological studies using

Drosophila melanogaster suggest that Parkin and

PINK1 are involved, through a common pathway, in

maintenance of mitochondrial function and that PINK1

acts upstream of Parkin.6,7 Thus, it is possible that

reduced activities of both gene products significantly

lower the threshold of nigral degeneration compared

with loss of activity of either Parkin or PINK1 alone.

In the present study, we screened for PINK1 muta-

tions in Parkinsonism patients with parkin mutations

and detected patients with both PINK1 and parkinmutations. Clinicogenetic analysis revealed that the

presence of PINK1 mutation in addition to parkinmutation could hasten the disease process.

PATIENTS AND METHODS

Subjects

This study was approved by the ethics review com-

mittee of Juntendo University School of Medicine. All

subjects gave informed and written consent before par-

ticipation. We selected patients with one- (single heter-

ozygous, n 5 19; 19 probands), and two- (homozygous

or compound heterozygous, n 5 156; 119 probands)

parkin mutation(s). All patients were screened for par-kin mutations by PCR, direct sequencing, and gene

dosage analyses of all exons. The mean age at onset

was 40.6 6 17.6 years (6SD, range 18–75; one parkinmutation) and 27.9 6 9.9 years (range 6–61; two par-kin mutations). Among the total of 175 patients, 130

(74.3%) had family histories of Parkinsonism, and 149

(85.1%) were Asian (133 Japanese, 6 Chinese, 6 Ko-

rean, and 4 Taiwanese). The remaining were 15 Isra-

elis, 3 Americans, 2 Tunisians, 2 Greeks, 1 Canadian,

1 German, 1 Iraqi, and 1 Moroccan.

Genetic Analyses

Genomic DNA samples were sequenced for all

exons and splice junctions of PINK1 using BigDye

Terminator v1.1 Cycle Sequencing kit and 3130

Genetic Analyzer (Applied Biosystems, Foster City,

CA). Only patients with heterozygous PINK1 mutation

were also screened by gene dosage analyses of all

exons of PINK1 by real-time PCR using TaqMan

probes and ABI PRISM 7700 Sequence Detector

(Applied Biosystems). Microsatellite markers flanking

PARK2 and PARK6 loci were genotyped by PCR

using fluorescence labeled primers, 3130 Genetic Ana-

lyzer, and GeneMapper software (Applied Biosystems).

PCR, sequencing, and real-time PCR were used stand-

ard methods and published primers and probes.8

RESULTS

We identified a novel heterozygous mutation

(p.R58-V59insGR) in exon 1 of PINK1 in a pair of

Japanese siblings with homozygous parkin mutations

(p.T175PfsX2: Fig. 1; Family A, A3 and A4). These

mutations were absent in 300 Japanese normal chromo-

somes, indicating that the mutations might be patho-

genic. We also detected the same heterozygous PINK1mutation in one of the unaffected parents who had het-

erozygous parkin p.T175PfsX2 mutation (Fig. 1; Fam-

ily A, A1). Another heterozygous PINK1 mutation

(p.R407Q) in exon 6 was detected in a pair of Chinese

siblings with compound heterozygous parkin muta-

tions (p.C441R and p.A138GfsX7: Fig. 1; Family C).

The p.R407Q mutation of PINK1 was reported previ-

ously in one Taiwanese patient with PD, but was

absent in 188 Taiwanese control chromosomes.9 We

1462 M. FUNAYAMA ET AL.


did not detect this mutation in 300 Japanese normal

chromosomes.

Next, we screened mutations of PINK1 in patients

who had heterozygous parkin mutation. We detected a

patient with sporadic PD with heterozygous PINK1mutation (p.E476K) and heterozygous parkin mutation

(p.P437L: Table 1; Patient D), which were absent in

300 Japanese normal chromosomes. In addition, we

performed gene dosage analyses of PINK1 for subjects

who were identified with a single heterozygous muta-

tion of the gene. No exonic rearrangements in PINK1were detected in any of the subjects.

We found one patient (Patient B1) from the original

sample series who had homozygous parkin p.T175

PfsX2 mutation (the same mutation in Patients A3 and

A4) but no PINK1 mutation. Haplotype analyses of

PARK2 and PARK6 loci in families A and B revealed

a common haplotype in PARK2, but not in PARK6

locus (Fig. 1). The p.T175PfsX2 mutation was absent

in 108 normal chromosomes from the Kyusyu region

in Japan (families A and B originated from Kyusyu

region). These results suggest that p.T175PfsX2 muta-

tion of parkin spread from a single founder. With

regard to the clinical features, the age at onset in

patients of family A who had both homozygous parkinmutation (p.T175PfsX2) and heterozygous PINK1mutation (p.R58-V59insGR) was more than 10 years

earlier than that in Patient B1 who had only homozy-

gous parkin mutation (Table 1). In addition, the age at

onset was significantly lower in patients with both two

parkin and one PINK1 mutations (Patients A3, A4, C1,

and C2) compared with the only two parkin mutations

(P 5 0.025, Student’s t-test). Interestingly, two of the

three patients with PD of family A had nondrug-

FIG. 1. Pedigrees of families analyzed in this study. Solid bars indicate shared disease haplotype. DNA of Patient A2 was not available. The hap-lotypes with undetermined phases in proband B-B1 are shown in parentheses. Int, intron.



induced schizophrenia with hallucination. None of the

patients in this cohort other than family A had schizo-

phrenia. In addition, Patient B1 had hallucination and

Patient D had depression.

DISCUSSION

In the present study, we set out to investigate

whether Parkin and PINK1 could influence each other

in patients with PD, based on the reports that Parkin

and PINK1 share a common pathway using Drosophilamodels.6,7 We identified digenic mutations of parkinand PINK1 and found that PINK1 mutation could

modify the clinical course of parkin mutation-positive

parkinsonism. Our results suggest that a single hetero-

zygous mutation of PINK1 might act not only as a

susceptibility gene3 but also as a modifier gene, in the

pathogenesis of PD.

The relatively high frequency of PINK1 heterozy-

gous mutation identified in the present study (2.2% in

PD vs. 0% in controls) is similar to that reported in a

recent study (1.2% in PD vs. 0.4% in controls).3 These

results suggest that PINK1 heterozygous mutation

might also increase the risk of development of PD in

patients who have mutations in other PD genes. Con-

sidering Patient D (Table 1), heterozygous PINK1p.E476K mutation was reported previously in three

patients and two control subjects.3,10 In addition, heter-

ozygous p.P437L of parkin was found at the same fre-

quency in patients and control subjects,11 whereas

none of Japanese 300 normal chromosomes harbored

these mutations in the present study. This could repre-

sent differences based on ethnicity. Observation of

patients carrying single nucleotide polymorphisms in

both parkin and PINK1 might be somewhat related to

the position of mutated amino acids, the type of muta-

tion, and one or more of the other gene mutations. On

the other hand, the presence of asymptomatic carrier

with the digenic mutations (family A-A1) also indi-

cates the role of heterozygous mutation of PINK1 in

disease modification and suggests that other factors

such as aging and environment are required for the de-

velopment of the disease.

Based on recent reports, asymptomatic carriers of

heterozygous parkin or PINK1 mutations exhibit low18F-dopa uptake in the putamen on positron emission

tomography.12,13 These studies suggest that heterozy-

gous mutation of parkin or PINK1 gradually impairs

the function of dopaminergic neurons. Interestingly,

our patients of Family A, B, and D also developed

TABLE 1. Clinical features of patients

Patient A2 A3 A4 B1 C1 C2 DOrigin Japan Japan Japan Japan Hong Kong Hong Kong Morocco

Age at onset 15 12 20 30 18 18 35Disease duration 38 25 9 36 22 17 18Sex M F F F M F MResting tremor 1 1 1 2 2 2 1Rigidity 1 1 1 NA 1 1 1Bradykinesia 1 1 2 1 1 1 1Postural instability 1 1 2 2 1 1 2Frozen gait 2 1 2 1 2 2 1Clinical response to levodopa 1 1 1 1 1 1 1Wearing off 2 1 2 1 1 1 1On off 2 1 2 1 1 1 1Asymmetry at onset 1 1 1 2 2 2 1Incontinence 2 2 2 2 2 2 2Urinary urgency 2 2 2 NA 2 2 1Levodopa-induced dyskinesia 1 1 2 1 NA 1 1Sleep benefit 2 2 2 1 1 1 NADystonia at onset 2 1 2 2 1 1 1Hyperreflexia 2 2 2 2 1 1 2Dementia 2 1 2 NA 2 2 2Depression 2 2 2 2 2 2 1Hallucination 1 1 2 1 2 2 2UPDRS III (on/off) 20/NA 32/NA NA 15/34 NA NA NAOther psychosis sch sch 2 2 2 2 2Special comment 2 2 2 2 2 2 RLS, RBD, facial dyskinesia

with grimacing, severedysarthria from onset

sch, schizophrenia; UPDRS, unified Parkinson’s disease rating scale (motor score) in on and off condition; NA, not applicable or not available;RLS, restless legs syndrome; RBD, REM sleep behavior disorder; 1, present; 2, absent.

1464 M. FUNAYAMA ET AL.


psychiatric disorders. Previous studies also reported

that some parkin and PINK1 mutations, even though

heterozygous mutations, could be related to levodopa-

responsive parkinsonism and psychiatric clinical pic-

tures.1,4,5 In this regard, our results might further indi-

cate that parkin and PINK1 mutations could be

involved in psychiatric disorders not only singularly

but also in combination. Furthermore, additional heter-

ozygous PINK1 mutation could hasten the age at onset

of the disease. Combining the previous reports, our

results emphasize that some heterozygous PINK1mutations might be related to the development of

PD.3,10 However, further genetic and functional analy-

ses are required before one can make definite

conclusions.

Intriguingly, digenic mutations of PINK1-DJ-1 and

parkin-LRRK2 have recently been reported.14,15

Screening for digenic or more mutations in responsible

genes for familial PD could lead to the elucidation of

the molecular pathway involved in nigral degeneration.

In this regard, the mitochondrion is a good target for

elucidating the pathogenesis of PD since Parkin,

PINK1, and DJ-1 could be related to the mitochondrial

function/dysfunction. Indeed, several studies high-

lighted the role of ARP gene products in maintaining

mitochondrial function and in the pathogenesis of PD.

Our results and these findings suggest that, multigenic

mutation screening and analyses for interactions among

related gene products could help enhance our under-

standing of the pathogenesis of PD.

Acknowledgments: This study was supported by a grantfrom the Japanese Ministry of Education, Culture, Sports,Science and Technology (to NH, Grant-in-Aid for ScientificResearch No. 17390256), and Health and Labour SciencesResearch Grant from the Japanese Ministry of Health, Labourand Welfare (to NH, No. 2080218510).

REFERENCES

1. Kubo S, Hattori N, Mizuno Y. Recessive Parkinson’s disease.Mov Disord 2006;21:885–893. Review.

2. Sun M, Latourelle JC, Wooten GF, et al. Influence of hetero-zygosity for parkin mutation on onset age in familial Parkinsondisease: the GenePD study. Arch Neurol 2006;63:826–832.

3. Abou-Sleiman PM, Muqit MM, McDonald NQ, et al. A heterozy-gous effect for PINK1 mutations in Parkinson’s disease? AnnNeurol 2006;60:414–419.

4. Steinlechner S, Stahlberg J, Voelkel B, et al. Co-occurrenceof affective and schizophrenia spectrum disorders with PINK1mutations. J Neurol Neurosurg Psychiatry 2007;78:532– 535.

5. Ephraty L, Porat O, Israeli D, et al. Neuropsychiatric and cognitivefeatures in autosomal-recessive early parkinsonism due to PINK1mutations. Mov Disord 2007;22:566–569.

6. Park J, Lee SB, Lee S, et al. Mitochondrial dysfunction in Drosoph-ila PINK1 mutants is complemented by parkin. Nature 2006;441:1157–1161.

7. Clark IE, Dodson MW, Jiang C, et al. Drosophila pink1 is requiredfor mitochondrial function and interacts genetically with parkin.Nature 2006;441:1162–1166.

8. Hatano Y, Li Y, Sato K, et al. Novel PINK1 mutations in early-onset parkinsonism. Ann Neurol 2004;56:424–427; Erratum in:Ann Neurol 2004;56:603.

9. Fung HC, Chen CM, Hardy J, Singleton AB, Lee-Chen GJ, WuYR. Analysis of the PINK1 gene in a cohort of patients with spo-radic early-onset parkinsonism in Taiwan. Neurosci Lett 2006;394:33–36.

10. Zadikoff C, Rogaeva E, Djarmati A, et al. Homozygous and hetero-zygous PINK1 mutations: considerations for diagnosis and care ofParkinson’s disease patients. Mov Disord 2006;21:875–879.

11. Kay DM, Moran D, Moses L, et al. Heterozygous parkin pointmutations are as common in control subjects as in Parkinson’spatients. Ann Neurol 2007;61:47–54.

12. Khan NL, Valente EM, Bentivoglio AR, et al. Clinical and subclini-cal dopaminergic dysfunction in PARK6-linked parkinsonism: an18F-dopa PET study. Ann Neurol 2002;52:849–853.

13. Khan NL, Scherfler C, Graham E, et al. Dopaminergic dysfunctionin unrelated, asymptomatic carriers of a single parkin mutation.Neurology 2005;64:134–136.

14. Tang B, Xiong H, Sun P, et al. Association of PINK1 and DJ-1 con-fers digenic inheritance of early-onset Parkinson’s disease. HumMol Genet 2006;15:1816–1825.

15. Dachsel JC, Mata IF, Ross OA, et al. Digenic parkinsonism: investi-gation of the synergistic effects of parkin and LRRK2. NeurosciLett 2006;410:80–84.



Distribution of Motor ImpairmentInfluences Quality of Life in

Parkinson’s Disease

Kim C. Stewart, MS,1 Hubert H. Fernandez, MD,2,3

Michael S. Okun, MD,2,3 Charles E. Jacobson, BS,3

and Chris J. Hass, PhD1,2*

1Department of Applied Physiology and Kinesiology,University of Florida, Gainesville, Florida, USA; 2Movement

Disorders Center, University of Florida,Gainesville, Florida, USA; 3Department of Neurology,

University of Florida, Gainesville, Florida, USA

Abstract: We evaluated the relationship between upper ex-tremity (UE) and lower extremity (LE) motor impairmentsin Parkinson’s disease (PD) to overall disability and qualityof life (QoL) measures. A total of 639 patients who werediagnosed with idiopathic PD were administered the Uni-fied Parkinson’s Disease Rating Scale (UPDRS), QoL,activities of daily living (ADL), and behavioral scales.Composite UE and LE scores from the motor section ofthe UPDRS were correlated with ADL, QoL, and behav-ioral measurement scores while controlling for disease du-ration. Patients with greater UE and LE motor impair-ments had lower QoL scores. However, LE impairmentshad a greater influence than UE impairments across allQoL measures. � 2008 Movement Disorder Society

Key words: quality of life; upper and lower extremityimpairments

Parkinson’s disease (PD) is a progressive neurodege-

nerative disorder that affects motor, behavioral and

cognitive functioning, and subsequently it affects the

quality of life (QoL). The Unified Parkinson’s Disease

Rating Scale (UPDRS) is the gold standard for evaluat-

ing motor symptoms and for assessing disease progres-

sion overtime, while the PD Questionnaire-39 (PDQ)

has been useful in quantifying both activities of daily

living (ADL) and behavioral/psychological QoL.1,2

Although an increasing number of researchers have

investigated the impact of motor and nonmotor symp-

toms on QoL,2–6 to the best of our knowledge, none

have investigated the influence of location or distribu-

tion of motor impairments. This finding is interesting

because several reports suggest that loss of dexterity

[upper extremity (UE) control] and gait speed [lower

extremity (LE) control] are both independent and

strong indicators of loss of independence.4,7 We there-

fore evaluated the influence of distribution (i.e. UE vs

LE impairment) on several PD motor and behavioral

QoL outcome measures. We hypothesized that UE

impairment that is heavily responsible for dexterity-de-

pendent daily activities will have a stronger influence

on motor QoL measures, whereas LE impairment, pos-

sibly the single best indicator of autonomy and inde-

pendence would have a stronger influence on behav-

ioral QoL.


A total of 639 participants diagnosed with idiopathic

PD by a fellowship-trained movement disorders spe-

cialist (using the United Kingdom Brain Bank Criteria

for Parkinson’s Disease8) at the University of Florida

Movement Disorders Center between 2002 and 2006

were prospectively administered motor, QoL, and be-

havioral scales. Specifically, the UPDRS parts I, II,

and III (in the ‘‘off’’ medication state), PDQ-39, modi-

fied Hoehn and Yahr (H&Y) Staging, Beck’s Depres-

sion Inventory (BDI), and the Schwab and England

(S&E) Disability Scale (in the ‘‘on’’ medication state)

were administered to all subjects. The PDQ-39 was

delineated and motor and behavioral QoL sub scores

were generated by combining the questions that are

related to UE and LE motor activities (1–16) and ques-

tions that referred to behavioral/psychological measures

(17–39), respectively. To partial out the influence of

UE and LE impairment, we computed a ‘‘percent

impairment score’’ that was determined by dividing

each cumulative score from the UE and LE motor

items to its corresponding highest possible score from

the UPDRS motor subsection and multiplying by 100.

These scores were then compared with the motor and

behavior scale scores previously mentioned using

Spearman’s bivariate correlations. Partial correlations

were also carried out to account for the influence of

disease duration. Linear regression models with

UPDRS UE and LE as the predicting variables and dis-

ease duration as a controlling covariate were developed

to determine the combined variance and strength of

prediction on each motor and behavioral measure that

demonstrated a statistically significant (P < 0.05) and

moderate (r > 0.3) or strong (r > 0.5) correlation.

*Correspondence to: Chris Hass, Department of Applied Physiol-ogy and Kinesiology, University of Florida, Florida Gym Room 151,Gainesville, Florida 32611. E-mail: [email protected]

Received 15 January 2008; Revised 10 April 2008; Accepted 6May 2008



1466 K.C. STEWART ET AL.


RESULTS

Our cohort of 639 PD patients had a mean: age of

66.83 6 11.22 years, disease duration of 8.56 6 7.51

years, and UPDRS motor ‘‘off’’ score 39.98 6 13.77.

Table 1 displays the mean and standard deviations of de-

mographic data, including the modified H&Y stage dis-

tribution, and the impairment and disability measures.

Overall, both the UE and LE impairment scores

revealed meaningful correlation to all ADL measures

(i.e. PDQ motor composite score, S&E Disability

Scale) and moderate correlations to behavioral QoL

measures (i.e. PDQ behavioral composite scores,

UPDRS part I, and BDI scores). Even when accounting

for disease duration, similar results were observed

(Table 2). Of interest, the LE motor impairment com-

posite score had a stronger correlation magnitude for

each motor and behavioral measure when compared

with the UE motor impairment composite score.

Regression analyses identified that LE motor impair-

ment had a stronger influence on all motor and behav-

ioral QoL measures when compared with the UE motor

impairment (Table 2).

DISCUSSION

Our findings confirm previous investigations, which

observed that greater motor impairments were associ-

ated with lower ADL and QoL scores.4–6 However,

our data revealed that, contrary to our original hypoth-

esis, both motor and behavioral QoL measures were

more strongly correlated with LE impairments than UE

impairments. Thus, in this large and diverse cohort of

PD patients, it appears that loss of LE function has

more detrimental effects on self-reported motor and

psychological QoL measures than UE disability. These

results are supported by previous findings that gait dis-

orders,3 postural instability,5 and freezing4 strongly

influence QoL. Because of fear of falling or being

unable to move about independently, patients tend to

avoid participating in activities in public and many

social events possibly leading to poorer QoL.

Other important aspects that may contribute to the

lesser impact of the UE impairments on ADL and QoL

measures include the use of compensatory strategies and

the general domain of the UPDRS motor questionnaire.

For example, compensation of skills via the less affected

arm/hand may possibly allow for completion of ADL’s;

however, the less affected leg may not easily compen-

sate for ADL’s such as walking, stair, climbing, etc.

Also, the general domains of the items assessed in the

motor section of the UPDRS may influence the results.

Impairment in UE motor items (such as finger taps) may

be less reflective of UE ADLs (such as eating or dress-

ing) compared with LE motor items, which may more

directly assess gait and postural stability. Future studies

may prove that items relating more to ADL will have a

stronger effect on perceived QoL.

Previous studies suggest that depression may be the

main determinant of poor QoL scores in PD.2 Although

motor impairment was not significantly related to

TABLE 1. Mean and standard deviations of demographicdata and QoL questionnaire scores

Demographics/QoL Scores Mean 6 SDHoehn-Yahr

staging Mode

Age 66.83 6 11.22Disease duration (in yr) 8.56 6 7.51 Stage 1 8UPDRS motor total 39.98 6 13.77 Stage 1.5 19aPDRS UE (% score) 47.24 6 15.61 Stage 2 139aUPDRS LE (% score) 26.81 6 12.77 Stage 2.5 114aPDQ-39 Motor 42.13 6 26.37 Stage 3 188aSchwab and England

(investigator)73.77 6 19.38 Stage 4 71

aSchwab and England(patient)

74.69 6 19.31 Stage 5 33

bDQ-39 Behavior 27.31 6 15.33

aMotor and activities of daily living measures.bBehavioral and psychological measures.UE, upper extremity; LE, lower extremity; PDQ, Parkinson’s Disease

Questionnaire; UPDRS, Unified Parkinson Disease Rating Scale.

TABLE 2. UE and LE UPDRS correlations to all QoL measures and regression model R2, b,and P value for each outcome measure that had a moderate to strong correlation

N 5 639

Schwab and Englandinvestigator,R2 5 0.195

Schwab and Englandpatient,

R2 5 0.479PDQ motor,R2 5 0.464

PDQ behavior,R2 5 0.446

Beck’sdepression

indexUPDRSpart I

UE UPDRS r 5 20.53 r 5 20.49 r 5 0.49 r 5 0.32 r 5 0.19 r 5 0.26b 5 0.05 (P 5 0.37) b 5 0.06 (P 5 0.28) b 5 0.03 (P 5 0.48) b 5 20.04 (P 5 0.49) – –

LE UPDRS r 5 20.69 r 5 20.64 r 5 0.66 r 5 0.42 r 5 0.29 R 5 0.35

b 5 20.65 (P < 0.001) b5 0.64 (P < 0.001) b 5 0.61 (P < 0.001) b 5 0.42 (P < 0.001) – –

All correlations were significant at P < 0.001.Bold font 5 moderate (r > 0.3) or strong (r > 0.5) correlation.Each regression model included disease duration as a controlling covariate.UE, upper extremity; LE, lower extremity; PDQ, Parkinson’s Disease Questionnaire; UPDRS, Unified Parkinson Disease Rating Scale.

1467QUALITY OF LIFE IN PARKINSON’S DISEASE


depression scores in this large cohort, both UE and LE

impairment influenced measures of QoL. This finding

suggests that the relationship between motor impair-

ment and depression ratings is complicated and their

singular and combined effects on QoL warrant further

large-scale study.

Although data were obtained from a very large sam-

ple of patients representing a wide spectrum of disease

severity, this study does possess certain limitations.

The motor evaluations were obtained in the ‘‘off’’ state

to obtain true motor severity and to minimize the vary-

ing influence of medications. The behavioral measures,

however, were obtained while patients where in the

‘‘on’’ state. This incongruence may influence the rela-

tionship between motor impairment and QoL. Subse-

quent studies should examine the LE and UE influence

on QoL in corresponding ‘‘off’’ as well as ‘‘on’’ states.

In summary, significant correlations between motor

impairments from the UPDRS and other ADL and

QoL measuring systems exist and show a greater LE

influence on both motor and behavioral measures. On

the basis of these results, we suggest that therapists

and caregivers should learn to recognize potential UE

and LE mismatches in motor dysfunction and in its

impact on QoL.

Acknowledgments: This study was funded in part by agrant received by the University of Florida Parkinson’s Dis-ease and Movement Disorders Center as National Parkinson’sFoundation Center of Excellence.

REFERENCES

1. Peto V, Jenkinson C, Fitzpatrick R, Greenhall R. The develop-ment and validation of a short measure of functioning and wellbeing for individuals with Parkinson’s disease. Qual Life Res1995;4:241–248.

2. Schrag A. Quality of life and depression in Parkinson’s disease.J Neurol Sci 2006;248:151–157.

3. Gomez-Esteban JC, Zarranz JJ, Lezcano E, et al. Influence ofmotor symptoms upon the quality of life of patients with Parkin-son’s disease. Eur Neurol 2007;57:161–165.

4. Moore O, Peretz C, Giladi N. Freezing of gait affects quality oflife of peoples with Parkinson’s disease beyond its relationshipswith mobility and gait. Mov Disord 2007;22:2192–2195.

5. Schrag A, Jahanshahi M, Quinn N. What contributes to quality oflife in patients with Parkinson’s disease? J Neurol NeurosurgPsychiatry 2000;69:308–312.

6. Schrag A, Jahanshahi M, Quinn N. How does Parkinson’s diseaseaffect quality of life? A comparison with quality of life in thegeneral population. Mov Disord 2000;15:1112–1118.

7. Bloem BR, Hausdorff JM, Visser JE, Giladi N. Falls and freezingof gait in Parkinson’s disease: a review of two interconnected,episodic phenomena. Mov Disord 2004;19:871�884.

8. Hughes AJ, Ben-Shlomo Y, Daniel SE, Lees AJ. What featuresimprove the accuracy of clinical diagnosis in Parkinson’s disease:a clinicopathologic study. Neurology 1992;42:1142�1146.

A Previously Undiagnosed Caseof Gerstmann-Straussler-ScheinkerDisease Revealed by PRNP Gene

Analysis in Patients withAdult-Onset Ataxia

Claudia Cagnoli, PhD,1 Alessandro Brussino, MD,1

Luca Sbaiz, PhD,2 Eleonora Di Gregorio, MSc,1

Cristiana Atzori, MSc,3 Paola Caroppo, MD,4

Laura Orsi, MD,4 Nicola Migone, MD,1 Carlo Buffa, MD,3

Daniele Imperiale, MD,3 and Alfredo Brusco, PhD1*

1Department of Genetics, Biology and Biochemistry,University of Torino, Medical Genetics Unit, Az. Osp.

San Giovanni Battista, Torino Italy; 2Azienda OspedalieraS. Anna di Torino, Italy; 3Centro Diagnosi Osservazione

Malattie Prioni D.O.M.P. - A.S.L.3, Torino, Italy; 4S.C.D.U.Neurologia I, Dipartimento di Neuroscienze, Az. Osp.

San Giovanni Battista, Torino, Italy

Abstract: Ataxia is a frequently reported symptom inprion diseases (PD) and it is characteristic of Gerstmann-Straussler-Scheinker syndrome (GSS), a genetic PDmainly related to the P102L mutation in the PRNP gene.Our aim was to screen for the P102L and other six knownPRNP gene mutations (P105L, A117V, Y145X, D202N,E200K, and V210I) a group of 206 consecutive patientsdiagnosed with adult-onset cerebellar ataxia of unknownorigin. The patients, negative for the most commonacquired and genetic forms, were analyzed using a combi-nation of restriction endonuclease digestion and pyrose-quencing; eight, affected by ataxia and cognitive dysfunc-tion, were also sequenced for the PRNP gene. One patientresulted to be heterozygous for the P102L mutation.Retrospectively, the clinical picture was consistent with a‘‘classical’’ GSS phenotype. In conclusion, the screeningfor the P102L mutation, or even the sequencing of thePRNP gene should be taken in consideration in patientswith late-onset ataxia (>50 years). � 2008 MovementDisorder Society

Key words: Gerstmann-Straussler-Scheinker syndrome;prion disease; PRNP; ataxia; dementia

Prion diseases (PD) are fatal neurodegenerative dis-

orders characterized by rapidly progressive dementia

associated with a variety of neurological signs.1 PD

Claudia Cagnoli and Alessandro Brussino contributed equally tothis work.

*Correspondence to: Alfredo Brusco, Department of Genetics,Biology and Biochemistry, University of Torino, via Santena, 19 -10126 Torino, Italy. E-mail: [email protected]

Received 4 September 2007; Revised 20 December 2007;Accepted 24 December 2007



1468 C. CAGNOLI ET AL.


may occur as sporadic, infective or genetic forms, the

latter being caused by mutations in the prion protein

coding gene (PRNP). Ataxia is frequently reported in

PD and it is characteristic of Gerstmann-Straussler-

Scheinker disease (GSS), an exceedingly rare PD

mainly related to the P102L mutation in the PRNPgene.2,3 The P102L GSS phenotype is characterized by

a progressive cerebellar syndrome with a median of

disease duration of 3.5 years (interquartile range 25

months), whereas cognitive symptoms are present only

in the latest stages.2 These data suggest the possibility

that isolated cases of GSS may be clinically classified

as sporadic ataxias. Sporadic ataxias are the less stud-

ied forms of ataxia, and only for a minority of patients

a mutation is found in the repeat-expanded SCA genes

or Friedreich ataxia gene.4,5 To our knowledge, there

are no literature reports on the occurrence of the

P102L and other PRNP gene mutations in patients

with sporadic ataxia.

Our aim was to verify if the P102L and other six known

mutations in the PRNP gene—four associated with a

GSS phenotype (P105L, A117V, Y145X, D202N) and

two with a Creutzfeldt-Jakob disease (CJD) phenotype

(E200K, V210I)—may account for a subset of patients

affected by adult-onset ataxia of unknown origin.2


Two hundred and six Italian unrelated adult pro-

bands (>30 years), consecutively referred to our labo-

ratory for SCA and/or Friedreich ataxia genetic testing,

from April 1997 to June 2005, were enrolled in this

study. All were negative for expansions at least in the

SCA1-3, 6, 7 and FXN loci. Men patients above 50

years were also excluded for FXTAS. Pathologic

expansions in the SCA17 gene were also ruled out in

patients with a dementia/cognitive impairment. Patients

with a positive history of alcohol abuse, cancer, or

chronic treatment with anticonvulsant drugs, or patients

with a clinical diagnosis of MSA were excluded by the

neurologists. One hundred sixty-two patients were iso-

lated cases, and forty-four showed a possible familiar-

ity (see Table 1). Genetic testing was performed after

informed consent.

Genomic DNA was isolated from total blood using

standard protocols (Qiagen, Mannheim, Germany). The

P102L mutation associated with GSS was tested using

AluI enzyme digestion: a 777 bp PCR product within

the coding region of the PRNP gene was amplified

using primers 50-gcagtcattatggcgaaccttg and 50-ccttcctcatcccactatcagg in a total volume of 25 lL, with2 mM MgCl2, 144 lM dNTPs, 400 nM of each primer,

1 U of Taq Gold (Applied Biosystems, Foster City,

CA). Cycling profile was: denaturation at 958C for 7

min; 40 cycles at 958C for 30 second, 588C for 30 sec-

ond, 728C for 1 min; and a final extension at 728C for

10 min. The PCR products (5 lL) were digested with

1 U of AluI (MBI, Vilnius, Lithuania) directly in the

PCR buffer, for 2 hours at 378C. Fragments were sepa-

rated on a 2% agarose-TBE 1X gel at 7.5 V/cm for

40 min, stained with ethidium bromide, and visualized

using a GelDoc XR apparatus (Bio-Rad Laboratories,

Hercules, CA). Expected bands were 361 and 416 bp

in normal samples, and 49, 312, and 416 bp in hetero-

zygous carriers of P102L.

For pyrosequencing a 395 bp fragment within the

coding region of the PRNP gene was amplified using

primers 50-biotin-gaggtggcacccacagtcag, and 50-ctga-gattccctctcgtactggg. The reaction volume of 50 lL con-

tained 2 mM MgCl2, 144 lM dNTPs, 200 nM for each

primer, 1 U of Taq Gold (Applied Biosystems). Cycling

profile was: denaturation at 958C for 7 min; 35 cycles

at 958C for 30 second, 608C for 30 second, 728C for

1 min; and a final extension at 728C for 10 min.

Sequencing primers, designed using a specific soft-

ware (primerdesign.pyrosequencing.com), were hybri-

dized to the single stranded PCR product purified as

described by the manufacturer (Pyrosequencing AB,

Uppsala, Sweden), and incubated in the pyrosequenc-

ing reaction mixture. Some of the tested mutations

could be detected using a common primer. Mutations

tested were P102L-P105L (50-atgtgcttcatgttggttt),

TABLE 1. Clinical features of the ataxic patients tested

Gender (Man/Woman) 105/101Age at onset mean 6 S.D. (range) 51.1 6 12.4 (30–76 yr)Ataxia associated signsUnknown 59Pure ataxia 82Spastic ataxia 26Spastic ataxia and neuropathy 2Epilepsy 1Epilepsy and nystagmus 1Neuropathy 6Nystagmus 6Nystagmus and pyramidal signs 2Parkinsonism 1Retinitis pigmentosa 1Tremor 9Tremor and neuropathy 1Cognitive impairment 8Psychosis 1

Total patients 206Genetic transmissionHereditary with uncertain trasmissiona 23Autosomal dominanta 21Sporadic 162

aTransmission inferred from clinical and anamnestic data.

1469GSS DISEASE


A117V (50-caccactgccccag), Y145X (50-tcacgatag-taacggtcctc), E200K-D202N (50-gcgctccatcatcttaac),and V210I (50-tgggtgatacacatctgc). Mutations A117V

and V210I were analyzed in multiplex. An automated

pyrosequencing instrument, PSQ (Pyrosequencing AB,

Uppsala, Sweden) was used for to perform genotyping.

Positive controls were run for each tested mutation.

Nine affected index cases reported with ataxia associ-

ated with dementia or cognitive impairment were fully

sequenced. The coding region of the PRNP gene

(NM_001080123.1; chr20:4615069-4630233) was amp-

lified using primers designed to flank exon 2 and intron-

exon boundaries PRN_F1 (50-ccattgctatgcactcattca)and PRN_R1 (50-gctggaaaaagattagaaagatgg). Amplified

products were purified and directly sequenced using the

Big Dye Terminator Cycle Sequencing kit ver.1.1 on an

ABI Prism 3100 Avant automatic sequencer (Applied

Biosystems).

RESULTS

Screening for PRNP Mutations in Ataxic Patients

Using pyrosequencing or a combination of pyrose-

quencing and restriction endonuclease digestion, we

examined 206 patients affected by pure or complex

forms of ataxia—most without familial history of dis-

ease—for the P102L and other six PRNP mutations

(P105L, A117V, Y145X, D202N, E200K, and V210I).2

We identified one patient carrier for the P102L muta-

tion (patient 5,854, Fig. 1). The patient was also

sequenced to confirm the mutation, and resulted homo-

zygous for the Met129 polymorphism.

Furthermore, we fully sequenced the PRNP gene in

eight patients with ataxia associated with cognitive

decline (Table 2). We did not find any possible muta-

tion, although in one case (patient D099) a substitution

c.-10-21G>A, 21 bp upstream of the exon 2 acceptor

splicing site was detected. This substitution is not

reported as a polymorphism. Anyway, bioinformatics

analysis does not show a change in the splice site

score, and the position is not evolutively conserved in

Primates.

Clinical Description of Patient 5854

At the age of 52 years, the patient underwent a neu-

rological examination for the onset of gait unbalance

and frequent falls. Her family (of sicilian origin) had

no remarkable clinical history. Her clinical records did

not refer any relevant pathology until this age. Neuro-

logical examination revealed cerebellar ataxia with

bilateral dysmetria and diffuse hyperreflexia. Extrapyr-

amidal signs, involuntary movements, and gaze abnor-

malities were absent. Cognitive functions were normal.

Neurophysiologic investigations (electroencephalo-

graphic and electroneurographic studies, multimodal

evoked potentials) and brain MRI were normal, and in

FIG. 1. Pyrograms showing the affected subject (5854) identified in this paper (panel A), a subject previously diagnosed with GSS P102L muta-tion, used as positive control (panel B) and a normal wild type subject (panel C). Black arrows indicate the profile of the nucleotide c.305: G/Ain panels A and B, and G/G in panel C. Below each panel the region of 15 bp sequenced by pyrosequencing is reported.

1470 C. CAGNOLI ET AL.


particular cerebellar structures seemed unaffected. CSF

was acellular, sterile with normal protein levels and

no oligoclonal IgG bands. Cianocobalamin, folates,

and tocopherol plasma levels were within the normal

range. Anti-gliadin and anti-endomisium antibodies

were absent. No pathologic expansion was found in the

SCA1-3, 6-8, 10, 12, 17, DRPLA, and Friedreich

ataxia (FXN gene) loci. The patient received a diagno-

sis of sporadic ataxia of unknown origin. The clinical

picture progressively worsened up to akinetic mutism,

and the patient died from broncopneumonia in 2003,

after five years of disease. Autopsy was not performed.

DISCUSSION

Sporadic late-onset ataxia occurs in about one in

10,000 adults. In most cases the precise etiology remains

unknown.4,5 Ruling out acquired conditions, a genetic

cause is to be considered even in the absence of a posi-

tive familial history. In this view, SCA and FA genetic

testing are usually included in the diagnostic work-up of

sporadic patients with adult-onset ataxia and FXTAS

may be suspected for male patients above 50 years at

onset.6 Genetic PD, in particular GSS, are rarely taken

into account, because of their very low incidence (1–10

in 100 millions per year),2 although their initial clinical

presentation may resemble sporadic ataxia.

The present work was aimed to evaluate the occur-

rence of PRNP mutations (in particular the P102L,

associated with the classical GSS phenotype) in a

subset of patients with late-onset ataxia of unknown

origin.3 Out of 206 screened patients, a case with a clini-

cal diagnosis of sporadic ataxia was found to be heter-

ozygous for the P102L mutation. Retrospectively, the

clinical course of this patient was compatible with the

classical description of GSS phenotype characterized

by predominant progressive ataxia, and cognitive

symptoms only in the very late phases.

Pyrosequencing allowed us a rapid and affordable

screening, and we suggest it as a valid alternative to

restriction digestion if a small subset of mutations is to

be screened in large surveys. Finally, sequence of the

entire PRNP gene may reveal new point mutations

associated with complicated forms of ataxia and cogni-

tive decline.

The identification of a GSS patient in our survey of

ataxic subjects, negative for known hereditary forms, sug-

gests that PD, in particular GSS, should be included in

the differential diagnosis of late onset ataxia (>50 years)

of unknown origin through the screening for P102L muta-

tion or even the sequencing of the PRNP gene.

Acknowledgments: This work was supported by Telethongrant GGP07110, Associazione ‘‘E.E. Rulfo per la GeneticaMedica’’, MURTS 60% to A.B. The ‘‘Centro Diagnosi Osser-vazione Malattie Prioni D.O.M.P. - A.S.L.3’’ is supported byRegione Piemonte—Ricerca Sanitaria finalizzata. We thankthe technical assistance of Dr. P. Pappi, the clinicians whoreferred us their patients, and Dr. Scaroima for providinghelpful clinical information.

REFERENCES

1. Gambetti P, Kong Q, Zou W, Parchi P, Chen SG. Sporadic andfamilial CJD: classification and characterisation. Br Med Bull 2003;66:213–239.

2. Kovacs GG, Puopolo M, Ladogana A, et al. Genetic prion disease:the EUROCJD experience. Hum Genet 2005;118:166–174.

3. Ghetti B, Tagliavini F, Takao M, Bugiani O, Piccardo P. Hereditaryprion protein amyloidoses. Clin Lab Med 2003;23:65–85, viii.

4. Abele M, Burk K, Schols L, et al. The aetiology of sporadic adult-onset ataxia. Brain 2002;125(Part 5):961–968.

5. Schols L, Szymanski S, Peters S, et al. Genetic background of ap-parently idiopathic sporadic cerebellar ataxia. HumGenet 2000;107:132–137.

6. Brussino A, Gellera C, Saluto A, et al. FMR1 gene premutationis a frequent genetic cause of late-onset sporadic cerebellar ataxia.Neurology 2005;64:145–147.

TABLE 2. Clinical and genetic data of the patients sequenced for the PRNP gene

Sample Onset Inheritance Ataxia Other symptoms Cognitive symptoms Codon 129a Variants identified

5494 58 yr Sporadic Yes Psychosis V/M8712 65 yr Sporadic Yes FTDb V/M9015 30 yr Sporadic Yes Muscular hypotrophy Dementia V/M9101 �65 yr Sporadic Yes Dementia V/MB516 �65 yr Sporadic Yes Dementia V/VB915 �55 yr Familialc Yes Dementia M/MC265 65 yr Familiald Yes Dementia M/MD099 �65 yr Sporadic Yes Head tremor Dementiae V/M c.-10-21G>Af

aIndicates the presence of a Methionine (M) or Valine (V) at codon 129.bFTD, Frontotemporal dementia.cThree sisters affected by similar symptoms.dFamiliarity for cognitive decline.eMental retardation.fThe reference sequence used was NM_001080123.1.

1471GSS DISEASE


A Novel Mutation of thee-Sarcoglycan Gene in a ChineseFamily with Myoclonus-Dystonia

Syndrome

Xue-Ping Chen,1 Yang-Wei Zhang,1

Shu-Shan Zhang,1 Qin Chen, MD,1

Jean-Marc Burgunder, MD,1,2

Shu-Hui Wu,1 Yuan Yang, MD,3

Zu-Ming Luo,1 and Hui-Fang Shang, MD1*

1Department of Neurology, West China Hospital, SichuanUniversity, Chengdu, SiChuan, China; 2Department of

Neurology, Inselspital, University of Bern, Bern, Switzerland;3Department of Medical Genetics,

West China Hospital, Sichuan University, Chengdu, SiChuan,China

Abstract: In a Chinese myoclonus-dystonia syndrome (MDS)family presented with a phenotype including a typicalMDS, cervical dystonia, and writer’s cramp, genetic anal-yses revealed a novel 662 1 1insG heterozygous mutationin exon 5 in the e-sarcoglycan (SGCE) gene, leadingto a frameshift with a down stream stop codon. LowSGCE mRNA levels were detected in the mutation car-riers by real-time PCR, suggesting that the nonsensemutation might interference with the stability of SGCEmRNA. This is the first report on Chinese with a SGCEmutation leading to MDS. Our data support the fact thatsame mutation of SGCE gene can lead to a varied pheno-type, even in the same family. � 2008 Movement DisorderSociety

Key words: myoclonus-dystonia syndrome; e-sarcoglycangene; gene mutation

Myoclonus-dystonia syndrome (MDS) is an autoso-

mal dominant inherited movement disorder character-

ized by involuntary jerks and dystonic movements and

postures.1 Onset of MDS is usually in childhood or

adolescent. Myoclonic jerks affect mainly neck, trunk,

and both arms, but barely the lower extremities. Dysto-

nia usually presents with spasmodic torticollis and

writer’s cramp. Rarely, however, dystonia may be the

only presentation of the disease. Psychiatric symptoms

including depression, anxiety, obsessive-compulsive

behavior, panic attack, and personality disorder have

been reported in MDS.2,3

Recently, mutations in the e-sarcoglycan (SGCE)gene have been found to be the major cause of MDS,

and a reduced penetrance has been recognized.4–6 The

SGCE gene spans about 70 kb genomic DNA and maps

to the human chromosome 7q21.7 The number of exons

can vary between 6, 11, or 12. The protein may consist

of 260, 437, 451, and 462 amino acids (UCSC build

March 2006: http://genome.ucsc.edu). The translated

full sequence protein comprises a signal sequence, a

large extracellular domain, a transmembrane domain,

and a short cytoplasmic domain.7 Up to date, different

mutations of the SGCE gene have been identified.2,3,8,9

The majority of mutations were found in exons 1–7, and

the most frequently reported one is a p.R102X reported

in nine unrelated families.2,3,8,9 In this study, we have

screened the SGCE gene in a Chinese family with MDS

and found a novel heterozygote mutation. In addition,

because a mutation of DYT1 gene was detected in one

MDS family,10 we have screened this gene as well.

CLINICAL DATA AND FAMILY STUDY

Clinical Data

This nonconsanguineous Chinese family (see Fig. 1)

consisted of three generations including 26 members.

The proband (III:1), his father (II:4), his younger half-

brother (III:3), his uncle (II:6), and the daughter of his

uncle (III:4) were symptomatic. The deceased grandfa-

ther (III:1) was reported to have upper limbs jerking

starting around 40. The proband (III:1) developed myo-

clonus jerks located in the arms at the age of 18, fol-

lowed by neck, trunk, and legs. A head tilt occurred at

the age of 19. Neurological examination revealed

myoclonus of arms, neck, and legs, head turn to the

left. He presented to our department at the age of 23

and was diagnosed as a MDS. He had a mild anxiety.

His psychiatric history and profile was otherwise nor-

mal. Serum ceruloplasmin, erythrocytes, immunologic

tests, his brain MRI, EEG, EMG, and SEP were nor-

mal. Mini mental status evaluation (MMSE) was nor-

mal.

His father (II:4) had mild head tilt at the age of 20

but without progression and without myoclonus. His

uncle (II:6) developed similar symptoms like the pro-

band at the age of 17. His younger half-brother (III:3)

had head tilt at the age of 18 without myoclonus. The

daughter of his uncle (III:4) had writer’s cramp at the

age of 12 without myoclonus. The myoclonus of the

*Correspondence to: Hui-Fang Shang, Department of Neurology,West China Hospital, Sichuan University, Chengdu Wai Nan GuoXue Xiang Nr. 37, 610041 Chengdu, SiChuan, China.E-mail: [email protected]

Received 31 July 2007; Revised 6 February 2008; Accepted 6 Feb-ruary 2008



1472 X.-P. CHEN ET AL.


proband (III:1) and his uncle (II:6) was responsive

to ethanol ingestion. No psychiatric history and psychi-

atric profile of his affected relatives were reported.

Anxiety scale and depression scale and MMSE were

normal. Brain MRI, EEG, serum ceruloplasmin, and

immunologic tests of his affected relatives were

normal.

Blood samples from the proband and all his affected

relatives were collected for genetic study after informed

consent. DNA samples from 50 Chinese healthy sub-

jects were processed in the same way. This study was

approved by the local ethics committee.

MOLECULAR GENETIC METHODS

Mutation Analysis

DNA isolation and PCR amplification for SCGEwere performed as previously described (GeneBank

accession no. NT_007933).11 Exon 10 was amplified

with newly designed primers in this study. Published

primer sequences were used for DYT110 (GeneBank

accession no. NT_029366). Direct sequencing was per-

formed using the BigDye Terminator Cycle Sequenc-

ing Kit running on an ABI37300 automated DNA-

sequencing system. Sequencing results were compared

with the sequences published in GeneBank, NCBI.

Quantitative Assessment of SGCE

mRNA by RT-PCR

RNA from venous whole blood of the proband

(III:1), his affected half-brother (III:3) and two healthy

controls was isolated using TRizol1 (Invitrogen Life

Technologies, Carlsbad, CA) following the manufac-

ture’s instructions. After reverse transcription was per-

formed using Transcriptor cDNA Synthesis Kit (Super-

Script, Takara), a 805 bp transcript spanning exons

3 and 7 was amplified with published primer.6 The

sequencing result of RT-PCR product was compared

with the sequence of SGCE mRNA (SGCE GeneBank

accession no. NM_001099400). To perform real-time

RT-PCR, the primers and probes of SGCE and b-actin(ACTB, as an endogenous control) were designed using

the program Primer Express 1.5 (Applied Biosystems;

ACTB GeneBank accession no. NM_001101). The

sequence of SGCE primers and probes were given as

follows: 50-TTCCCGTTGCCATATCAAG-30 (forward);

50-GGACATCTGCACCAACCAT-30(reverse);50-FAM-

ACATAAACGCCCTCCTTCAGGTCATT-ECLIPSE-30

(Probe); product length 206 bp spanning exons 5 and

6. The sequence of ACTB primers and probes was the

following: 50-AAGGCCAACCGCGAGAA-30(forward);50-CCTCGTAGATGGGCACA-30(reverse);50-FAM-TC-

AACACCCCAGCCATGTACGT-TAMRA-30(probe);product length 166 bp.

The SGCE and b-actin were amplified in a final vol-

ume of 25 lL containing 1 iQ Supermix reagent (Bio-

Rad, USA), 0.2 lM of each primer and Taqman probe,

2 lL cDNA. Experiments were performed in triplicates

for each sample and with a no-template control on an

iCycler iQ Detection System (Bio-Rad) according to

manufacturer’s protocols with 40 cycles as follows:

948C for 20 s, 568C for 30 s, and 728C for 30 s.

Threshold cycle (Ct, the number of cycles necessary to

obtain a threshold fluorescent signal) numbers were

determined and transformed using the [DELTA]Ct

methods. RT-PCR products were separated on a 2.5%

agarose gel with ethidium bromide staining to verify

the expected product sizes. The amount of the respec-

tive template was then determined by calculating the

mean Ct value. Values of SGCE are expressed as an

amount of each cDNA normalized to b-actin. Data are

given as mean and standard error of the mean (SEM).

t-Tests were applied to compare values. Significance

level was set to P < 0.05.

RESULTS

A novel heterozygote mutation (662 1 1insG) in

exon 5 was detected in the proband and all affected

individuals (Fig. 2A forward sequencing and 2B

reverse sequencing, respectively), while not found in

the 50 healthy controls (100 control chromosomes; Fig.

2C). This ‘‘G’’ insertion was confirmed by the sequenc-

ing result of RT-PCR product (Fig. 2D). This mutation

predicted to lead to a frameshift with a stop at 18 co-

dons in the downstream sequence. Two new polymor-

phisms in the intron of the SGCE gene were found:

FIG. 1. Pedigree of the family with MDS. Circles represent femalesubjects; squares represent male subjects; arrow indicates proband;squares and circles with a line above indicate examined clinically;filled squares and circles indicate affected; squares and circles withdiagonal slash indicate deceased; the individual pedigree number isbelow each symbol.

1473NOVEL MUTATION OF THE "-SARCOGLYCAN GENE


390 1 15 G>A and 1304 1 16 T>G (data not shown).

No mutation was detected in the DYT1 gene.

Total RNA from III:1, III:3, and two healthy con-

trols was reverse transcribed into cDNA. The amplified

real-time RT-PCR products showed the expected sizes

on agarose gel staining with ethidium bromide. Quanti-

tative determination of SGCE mRNA expression nor-

malized to b-actin displayed a significant reduction of

SGCE mRNA in the III:1 (0.199 6 0.022) and III:3

(0.200 6 0.030) by a RT-PCR analysis when com-

pared with that of control 1 (0.436 6 0.030) and con-

trol 2 (0.416 6 0.017) (Fig. 2E).

DISCUSSION

To our knowledge, this is the first report of MDS in

China. The clinical phenotype is quite similar to that

described in other ethnic groups. A novel heterozygous

mutation (662 1 1insG) in exon 5 of the SGCE gene

detected in the five affected members of the family

was not found in 50 healthy subjects. This is therefore

very improbable, that this variant is a polymorphism

found in a Chinese population. So far, more than 30

mutations including nonsense mutations, insertion, de-

letion, and missense mutations have been identified in

the SGCE gene in patients with MDS.2,3,8,9 Our finding

adds a further novel mutation to the list. The affected

individuals in three generations of the family and the

presence of the same mutation in the III:1 and III:3

who have the same father but two different mothers

suggest that the inheritance of the family is autosomal

dominant and is compatible with maternal imprinting.6

In addition, no mutation of DYT1 gene was found, sup-

porting that DYT1 gene does not cause MDS typical

phenotypes.12

Significant lower SGCE expression detected in leu-

kocytes with the mutation by quantitative RT-PCR

than that of normal ones indicates that the nonsense

mutation might interferences with the stability of

SGCE mRNA through well-known nonsense-mediated

decay mechanism.13 The mutation leads to an early

stop codon, which predicts the translation of a short-

ened protein containing only the signal and the first

portion of the extracellular domain. Such a protein

would have lack of function, because it could not be

integrated at the cytoplasmatic membrane, leading to a

destabilization of the dystrophin associated protein

complex within neurons expressing SCGE, as this has

been described in muscle after mutation of other sarco-

glycan subunits.14 This mutation might produce low

level of truncated protein through nonsense-medicated

decay of the mutant transcript.15

In the family, affected individuals presented with a

variant phenotype including a typical MDS, mild cer-

vical dystonia and writer’s cramp, which were previ-

ously reported.3 Our data confirm the variability of

the clinical presentation even in a single family with

the same mutation. Thus, it is worth to screen the

mutation of SGCE gene in a patient with focal dysto-

nia who has a positive family history of myoclonus

dystonia.

Acknowledgments: This work was supported by NationalScience Fund of China (No. 30400144).

FIG. 2. DNA sequence chromatograms of portion of SGCE exon 5 showing a heterozygous ‘‘G’’ insertion in the proband (A: forward sequence,arrow; B: reverse sequence) and the same region in healthy control (C: forward sequence). cDNA sequencing of SGCE gene spanning the muta-tion showing a ‘‘G’’ insertion (D: arrow showing the end of the exon5). E: Quantitative real-time RT-PCR result: SGCE mRNA expression ofpatients were significantly lower than that of the controls (*P < 0.05 vs. control). [Color figure can be viewed in the online issue, which is avail-able at www.interscience.wiley.com.]

1474 X.-P. CHEN ET AL.


REFERENCES

1. Gasser T. Inherited myoclonus-dystonia syndrome. Adv Neurol1998;78:325–334.

2. Zimprich A, Grabowski M, Asmus F, et al. Mutations in thegene encoding epsilon-sarcoglycan cause myoclonus-dystoniasyndrome. Nat Genet 2001;29:66–69.

3. Asmus F, Zimprich A, Du Tezenas M, et al. Myoclonus-dystoniasyndrome: epsilon-sarcoglycan mutations and phenotype. AnnNeurol 2002;52:489–492.

4. Klein C, Liu L, Doheny D, et al. Epsilon-sarcoglycan mutationsfound in combination with other dystonia gene mutations. AnnNeurol 2002;52:675–679.

5. Hedrich K, Meyer EM, Schule B, et al. Myoclonus-dystonia:detection of novel, recurrent, and de novo SGCE mutations. Neu-rology 2004;62:1229–1231.

6. Muller B, Hedrich K, Kock N, et al. Evidence that paternalexpression of the epsilon-sarcoglycan gene accounts for reducedpenetrance in myoclonus-dystonia. Am J Hum Genet 2002;71:1303–1311.

7. Ettinger AJ, Feng G, Sanes JR. Epsilon-sarcoglycan, a broadlyexpressed homologue of the gene mutated in limb-girdle muscu-lar dystrophy 2D. J Biol Chem 1997;272:32534–32538.

8. Asmus F, Salih F, Hjermind LE, et al. Myoclonus-dystonia dueto genomic deletions in the epsilon-sarcoglycan gene. Ann Neurol2005;58:792–797.

9. Tezenas MS, Clot F, Vidailhet M, et al. Epsilon sarcoglycanmutations and phenotype in French patients with myoclonic syn-dromes. J Med Genet 2006;43:394–400.

10. Leung JC, Klein C, Friedman J, et al. Novel mutation in theTOR1A (DYT1) gene in atypical early onset dystonia and poly-morphisms in dystonia and early onset parkinsonism. Neuroge-netics 2001;3:133–143.

11. Shang H, Clerc N, Lang D, Kaelin-Lang A, Burgunder JM. Clini-cal and molecular genetic evaluation of patients with primarydystonia. Eur J Neurol 2005;12:131–138.

12. Furukawa Y, Rajput AH. Inherited myoclonus-dystonia: howmany causative genes and clinical phenotypes. Neurology 2002;59:1130–1131.

13. Maquat LE. Nonsense-mediated mRNA decay: splicing, translationand mRNP dynamics. Nat Rev Mol Cell Biol 2004;5:89–99.

14. Lin S, Ramelli GP, Moser H, Gallati S, Burgunder JM. A novelinsert mutation in gamma-sarcoglycan gene leads to severe child-hood autosomal recessive muscular dystrophy. J Neurol 2002;249:1608–1611.

15. Esapa CT, Waite A, Locke M, et al. SGCE missense mutations thatcause myoclonus-dystonia syndrome impair epsilon-sarcoglycantrafficking to the plasma membrane: modulation by ubiquitinationand torsinA. HumMol Genet 2007;16:327–342.

Lack of Trigemino-CervicalReflexes in ProgressiveSupranuclear Palsy

Michelangelo Bartolo, MD,1,2* Mariano Serrao, MD,PhD,3,4,5 Armando Perrotta, MD,1,2,5 Cristina Tassorelli,

MD, PhD,1,2 Giorgio Sandrini, MD,1,2 andFrancesco Pierelli, MD3,5

1Department of Neurological Sciences, University of Pavia,Pavia, Italy; 2Neurorehabilitation Unit, IRCCS ‘‘C. Mondino

Institute of Neurology’’ Foundation, Pavia, Italy;3Rehabilitation Unit, ICOT-Polo Pontino, University of Rome‘‘La Sapienza’’, Italy; 4Rehabilitation Unit, Policlinico Italia,Rome, Italy; 5IRCCS Neuromed, Pozzilli (IS), University of

Rome ‘‘La Sapienza’’, Italy

Abstract: Trigemino-cervical reflexes (TCRs) are multisy-naptic neck muscle withdrawal responses that are clearlyidentifiable in humans. Mediated by neural circuits atbrainstem level, these reflex responses have been found tobe significantly impaired in patients with Parkinson’s dis-ease (PD), and it has been hypothesized that a degenera-tion of brainstem neural structures could play a role inthese abnormalities. Because extensive neuronal degenera-tion at brainstem level has been demonstrated in progres-sive supranuclear palsy (PSP), in this pilot study we eval-uated the TCR responses in 12 subjects with PSP, and in16 healthy controls. The TCRs were absent in 11 out ofthe 12 PSP patients while clear responses were evoked inall the healthy subjects. These findings indicate that PSPpatients are unable to react to the painful stimuli to theface, suggesting a generalized impairment of the brain-stem circuits mediating TCRs. � 2008 Movement DisorderSociety

Key words: trigemino-cervical reflexes; PSP; trigeminalreflex; neck muscles

Progressive supranuclear palsy (PSP) is a neurodege-

nerative syndrome characterized by bradykinesia, pos-

tural instability, falls, axial rigidity, and supranuclear

gaze palsy.1 Marked brainstem atrophy and degenera-

tion2,3 may be related to these main clinical character-

istics.4

Investigations of brainstem pathway excitability in

PSP using electrophysiological techniques5,6 have indi-

*Correspondence to: Dr. Michelangelo Bartolo, NeurorehabilitationUnit, IRCCS Neurological Institute Casimiro Mondino Foundation,via Mondino, 2-27100 Pavia, Italy.E-mail: [email protected]

Received 4 April 2007; Revised 11 February 2008; Accepted 19February 2008



1475LACK OF THE TRIGEMINO-CERVICAL REFLEXES IN PSP


cated a dysfunction of brainstem neuronal circuits at

several levels.6

The trigemino-cervical reflexes (TCRs) method is

simple and quick to perform and allows the functional

exploration of an extensive neural network in the

brainstem.7,8 TCRs are elicited by electrical stimulation

of the trigeminal nerve at the supraorbital notch and

recorded as early, low-threshold, oligo-synaptic responses

and late, high-threshold, multisynaptic responses from

neck muscles.8 The late response shows higher reproduci-

bility and is mediated via a long pathway in the brain-

stem.8,9 TCRs have a nocifensive function and may be

considered the electrophysiological counterparts of head

retraction reflexes (HRRs).7,9,10

Patients with Parkinson’s disease (PD), compared

with healthy subjects, showed abnormal TCRs, i.e.,

higher latency and lower amplitude.11 It was hypothe-

sized that these abnormalities could be explained by

the brainstem degeneration found in PD.11–13 Given

the marked atrophy and degeneration at brainstem level

observed in PSP patients,2,3 we hypothesized that these

patients may show an abnormal HRR response to pain-

ful stimuli to the face.

We investigated the electrophysiological pattern of

TCRs in patients with PSP and compared the results

with those obtained in healthy subjects and with vari-

ous clinical variables.


Patients

Twelve patients with PSP (five women, seven men;

mean age 69.6 6 6.9 years, range 52–77; disease dura-

tion 4.9 6 1.8 years, range 2–9; disease severity 42.6 6

13.2, range 28–72, on the motor part of UPDRS-III)

were enrolled (Table 1). All fulfilled the NINDS-SPSP

diagnostic criteria for clinically probable PSP14 (Table 1).

Clinical assessment and diagnosis were performed by two

clinicians (G.S., F.P.), both experts in extrapyramidal

disorders.

Healthy Subjects

Sixteen age-matched healthy subjects (seven women,

nine men; mean age 60.4 6 7.5 years, range 42–71; no

clinical or family history of neurological disorders)

formed the control group

The local ethics committee approved the study, and

informed consent to participate was obtained from all

participants.

Neurophysiological Procedures

The TCRs were recorded using electromyographic

equipment (Medelec Synergy EMG/EP Monitoring

Systems; Oxford Instruments Medical, Inc. U.K.) as

previously described.7 During the experimental session,

the patients were seated comfortably with their head

unsupported. Their movements were unrestricted. Sev-

eral head positions were assumed in order to minimize

the background EMG activity of the neck muscles,

which was continuously monitored by visual inspec-

tion. Percutaneous electrical stimulation of the supraor-

bital nerve was performed through the application of

bipolar surface electrodes to the supraorbital notch;

rectangular electrical shocks (duration 0.5 ms) were

used.

The individual pain threshold (PT) was determined

by applying a series of stimuli of increasing and

decreasing (in 2 mA steps) intensity. The series was

TABLE 1. Clinical findings in patients with progressive supranuclear palsy

Patient Age (yrs) Gender Disease duration (yrs) Stage* UPDRS III

Clinical features

gp pi ar bs de

1 66 F 2 III–IV 35 2 1 1/2 1 22 71 M 3 I–II 28 1 1 1 2 23 76 M 4 IV 41 1 1 1 1 1/24 77 M 7 III 33 1 1 2 1 15 75 M 5 IV 40 1 1 1 1 26 74 F 6 IV 36 1 1 1 1 27 65 F 6 V 72 1 1 1 1 28 75 F 9 V 48 1 1 1 1 19 52 F 4 V 61 1 1 1 1 1/210 70 M 5 V 52 1 1 1 1 111 66 M 4 III–IV 35 1 1 1 2 112 69 M 4 IV 31 1 1 1 1 1

*Hoehn and Yahr stage (Hoehn and Yahr, 1967).gp, gaze palsy; pi, postural instability; ar, axial rigidity; bs, bulbar signs; de, dementia; 1, present; 2, absent.

1476 M. BARTOLO ET AL.


repeated three times. The mean of the electrical inten-

sities perceived as painful by the subjects was taken as

the PT. To minimize habituation of the reflex responses,

an interstimulus interval of at least 20 s was used. The

electrical stimuli were delivered at multiples (1X, 2X,

3X) of the PT. If reflex responses were not recorded at

3XPT, the stimulation intensity was increased, in 5 mA

steps, up to the maximal intensity tolerated by the

patient. Electromyographic signals were recorded from

the site corresponding to the semispinalis capitis muscle

(SSC) by means of silver surface electrodes fixed at the

level of the C3 (active electrode) and C7 (reference elec-

trode) vertebrae on the midline.7,15 The active electrode

was moved around to locate the point at which the EMG

signal showed maximal amplitude in a series of physio-

logical muscle contractions.

In 2 patients, the reflex was also recorded through a

needle electrode inserted in the SSC.16 In a further two,

we also recorded EMG responses from the sternocleido-

mastoid muscle bilaterally. The filter band-pass was 30

to 3000 Hz. Sensitivity was 200 to 500 lV. The room

temperature was maintained at 22 to 248C. All the tests

were performed by the same expert electromyographer.

Data Analysis

At least 10 trials were recorded, and the EMG signal

was rectified and averaged in a time window of 100 ms

after the stimulus. Reflex onset/offset was taken as the

instant in which the amplitude of the EMG signal rose

above/returned below 30 lV. The area under the curve

(AUC) of the reflex was measured between these two

points. TCR persistence (ratio of number of TCR

responses elicited to number of stimuli delivered) was

calculated. The reflex was considered absent if, after

electrical stimulation, the amplitude of the EMG signal

in each trial, in the time window considered, was

smaller than 30 lV and no detectable response could

be identified visually.

Statistical Analyses

Comparing patients and controls, we applied the chi-

square test to evaluate the difference in the frequency

distribution of the presence and absence of the reflex,

and the Mann-Whitney test to compare, when possible,

mean values of the reflex parameters (PT, latency, area).

RESULTS

The patients’ clinical data are reported in Table 1.

The frequency distribution of the presence/absence of

TCRs differed significantly between the patients and

the controls (P < 0.01): reflexes (both early and late

responses) were absent in all the patients but one. In

this patient, TCRs were elicited in only 4/10 trials

(40% reflex persistence) with the stimulus delivered at

over 3XPT (35 mA). The reflex latency was 47.9 ms

FIG. 1. Absence (left side) and presence (right side) of trigemino-cervical reflexes (TCRs) after stimulation of the supraorbital nerve in a repre-sentative patient with PSP and in a healthy subject, respectively. Note, in the left graph, that sensitivity was enhanced (50 lV instead of 200 lV)to highlight the absence of reflex responses.

1477LACK OF THE TRIGEMINO-CERVICAL REFLEXES IN PSP


and the area was 0.3 mV ms. Conversely, TCRs were

recorded in all the controls and were elicitable in 8–

10/10 (80–100%) trials; the mean latency was 42.4 67.6 ms and the mean area was 3.8 6 1.2 mV ms.

No TCRs were recorded from the SSC in the 2

patients examined. Similarly, no reflex response was

recorded from the sternocleidomastoid muscle bilater-

ally in the 2 patients evaluated. Because of the absence

of TCRs in the PSP patients, no statistical analysis was

possible to compare clinical features and electrophysio-

logical data. There was a significant difference in PT

(12.9 6 4.2 mA vs. 8.2 6 2.7 mA; P < 0.05) between

the patients and the controls.

DISCUSSION

Our main finding was that TCRs were absent in the

PSP patients but clearly identifiable in all the controls

(see Fig. 1). In only one PSP patient was it possible, at

high stimulation intensity, to identify the presence of

TCRs; however, these responses were poorly reproduc-

ible and showed a low amplitude compared to those of

the healthy subjects. These data suggest that the brain-

stem neuronal circuit mediating the TCRs is exten-

sively impaired in PSP.

TCRs are polysynaptic, nocifensive brainstem

reflexes originating from the trigeminal nerve afferents

connected to cervical spinal motoneurons of the neck

muscles.7,17

Animal studies have shown that the reticulo-spinal

neurons play a role in mediating TCRs18–20 and are

important for transmitting descending commands from

the brainstem to the spinal cord and for the motor con-

trol of head and neck postures and movements.18,19,21

These reticulospinal pathways originate from several

mesencephalic and pontomedullary reticular nuclei

(i.e., spinal trigeminal nucleus, pedunculopontine nu-

cleus, reticular formation).8,22 Anatomopathological

studies have clearly demonstrated that the degenerative

process in PSP involves several nuclei of the mid-brain

and ponto-reticular formation.2,3,23 These lesions may

explain some of the clinical features of PSP24,25 and

the lack of TCR responses we observed.

A recent study revealed several abnormalities of TCR

responses in PD; however, the TCRs were elicitable in

all the patients.11 In our study, we were not only unable

to record any TCR response in 11 out of 12 patients, but

also failed to observe, visually, any mechanical response

of head and neck after high-intensity electrical stimula-

tion of the facial muscles. This suggests that the whole

brainstem circuit mediating TCRs/HRRs is disrupted

and that PSP patients lack both the capacity to react to

painful stimuli applied to the face, and control of head

and neck tone, movement and posture.

Because the reflex was absent in almost all the

patients, no correlation with clinical features could be

found. The one patient who did display the reflex (low

amplitude and persistence) did not really differ clinically

from the other patients. Despite the lack of correlation

with the clinical variables of PSP, it can still be hypothe-

sized that the absent TCRs and abnormal neck move-

ments, head/neck postures and axial/neck rigidity

observed in these patients share a common pathophysio-

logical mechanism in the brainstem. Investigation of a

very large sample, including forms of possible PSP,

might possibly disclose correlations with the clinical

features. Interestingly, the TCR shares some similarities

with the R2 component of the blink reflex (bilateral na-

ture, time progression of latencies, duration). Evidence

of abnormal blink reflex responses in PSP patients8 sug-

gests that the neuronal substrate mediating these facial

and neck muscle responses may be part of the anatomi-

cal and functional system known to control head, facial,

and neck protective responses, as observed in ani-

mals,26,27 and that this system is widely impaired in

PSP. Because the technique we used is easy and quick to

perform, TCRs could be a useful tool in evaluating

brainstem function in PSP. However, further studies are

needed to establish a role for this method in distinguish-

ing patients with PSP from those affected by other par-

kinsonian disorders.

Acknowledgment: The authors thank Catherine Wrenn forrevising the English.

REFERENCES

1. Nath U, Ben-Shlomo Y, Thomson RG, Morris HR, Wood NW,Lees AJ, Burn DJ. The prevalence of progressive supranuclearpalsy (Steele-Richardson-Olszewski syndrome) in the UK. Brain2001;124 (Part 7):1438–1449.

2. Jellinger KA, Blancher C. Neuropathology. In: Litvan I, Agid Y,editors. Progressive supranuclear palsy: clinical and researchapproaches. Oxford: Oxford University Press; 1992. p 44–88.

3. Aiba I, Hashizume Y, Yoshida M, Okuda S, Murakami N, Uji-hira N. Relationship between brainstem MRI and pathologicalfindings in progressive supranuclear palsy-study in autopsy cases.J Neurol Sci 1997;152:210–217.

4. Litvan I, Hutton M. Clinical and genetic aspects of progressivesupranuclear palsy. J Geriatr Psychiatry Neurol 1998;11:107–114.

5. Valls-Sole J. Neurophysiological characterization of parkinsoniansyndromes. Neurophysiol Clin 2000;30:352–367.

6. Valls-Sole J. Neurophysiological aids to the diagnosis of progressivesupranuclear palsy (PSP). Suppl Clin Neurophysiol 2006;58: 249–256.

7. Serrao M, Rossi P, Parisi L, et al. Trigemino-cervical-spinalreflexes in humans. Clin Neurophysiol 2003;114:1697–1703.

8. Di Lazzaro V, Guney F, Akpinar Z, et al. Trigemino-cervicalreflexes: clinical applications and neuroradiological correla-tions. Suppl Clin Neurophysiol 2006;58:110–119.

1478 M. BARTOLO ET AL.


9. Leandri M, Gottlieb A, Cruccu G. Head extensor reflexevoked by trigeminal stimulation in humans. Clin Neurophy-siol 2001; 112:1828–1832.

10. Sartucci F, Rossi A, Rossi B. Trigemino-cervical reflex inman. Electromyogr Clin Neurophysiol 1986;26:123–129.

11. Perrotta A, Serrao M, Bartolo M, et al. Abnormal head noci-ceptive withdrawal reaction to facial nociceptive stimuli inParkinson’s disease. Clin Neurophysiol 2005;116:2091–2098.

12. Braak H, Rub U, Sandmann-Keil D, et al. Parkinson’s dis-ease: affection of brainstem nuclei controlling premotor andmotor neurons of somatomotor system. Acta Neuropathol(Berl) 2000; 99:489–495.

13. Braak H, Del Tredici K, Rub U, de Vos RA, Jansen SteurEN, Braak E. Staging of brain pathology related to sporadicParkinson’s disease. Neurobiol Aging 2003;24:197–211.

14. Litvan I, Bathia KP, Burn DJ, et al; Movement Disorders So-ciety Scientific Issues Committee. Movement Disorders SocietyScientific Issues Committee report: SIC Task Force appraisalof clinical diagnostic criteria for Parkinsonian disorders. MovDisord 2003;18:467–486.

15. Ertekin C, Celebisoy N, Uludag B. Trigeminocervical reflexeselicited by stimulation of the infraorbital nerve: head retrac-tion reflex. J Clin Neurophysiol 2001;18:378–385.

16. Kramer M, Schmid I, Sander S, Hogel J, Eisele R, Kinzl L,Hartwig E. Guidelines for the intramuscular positioning ofEMG electrodes in the semispinalis capitis and cervicismuscles. J Electromyogr Kinesiol 2003;13:289–295.

17. Di Lazzaro V, Restuccia D, Nardone R, Tartaglione T, Quar-tarone A, Tonali P, Rothwell JC. Preliminary clinical observa-tions on a new trigeminal reflex: The trigemino-cervicalreflex. Neurology 1996;46:479–485.

18. Inagaki M, Takeshita K, Nakao S, Shiraishi Y, Oikawa T.An electrophysiologically defined trigemino-reticulo-facial path-way related to the blink reflex in the cat. Neurosci Lett1989;96:64–69.

19. Alstermark B, Pinter MJ, Sasaki S. Descending pathwaysmediating disynaptic excitation of dorsal neck motoneuronesin the cat: brain stem relay. Neurosci Res 1992;15:42–57.

20. Sessle BJ, Hu JW, Amano N, Zhong G. Convergence of cuta-neous, tooth pulp, visceral, neck and muscle afferents ontonociceptive and non-nociceptive neurones in trigeminal subnu-cleus caudalis (medullary dorsal horn) and its implications forreferred pain. Pain 1986;27:219–235.

21. Drew T, Rossignol S. Functional organization within the med-ullary reticular formation of intact unanesthetized cat. I.Movements evoked by microstimulation J Neurophysiol1990;64:767–781.

22. Lee MS, Rinne JO, Marsden CD. The pedunculopontine nu-cleus: its role in the genesis of movement disorders. YonseiMed J 2000;41:167–184.

23. Josephs KA, Dickson DW. Diagnostic accuracy of progres-sive supranuclear palsy in the Society for ProgressiveSupranuclear Palsy brain bank. Mov Disord 2003;18:1018–1026.

24. Osaki Y, Ben-Shlomo Y, Lees AJ, Daniel SE, Colosimo C,Wenning G, Quinn N. Accuracy of clinical diagnosis ofprogressive supranuclear palsy. Mov Disord 2004;19:181–189.

25. Rivest J, Quinn N, Marsden CD. Dystonia in Parkinson’sdisease, multiple system atrophy, and progressive supranu-clear pasly. Neurology 1990;40:1571–1578.

26. Sumino R, Nozaki S. Trigemino-neck reflex: its peripheraland central organization. In: Anderson DJ, Matthews B,editors. Pain in the Trigeminal Region. Amsterdam: ElsevierNorth-Holland Biomedical Press; 1977. p 369–374.

27. Abrahams VC, Kori AA, Loeb GE, Richmond FJR, RosePK, Keirstead SA. Facial input to neck motorneurons: tri-gemino-cervical reflexes in the conscious and anaesthetizedcat. Exp Brain Res 1993;97:23–30.

Evaluating Parkinson’s DiseasePatients at Home: Utility of Self-Videotaping for Objective Motor,

Dyskinesia, and ON–OFFAssessments

Christopher G. Goetz, MD,1* Sue Leurgans, PhD,1

Vanessa K. Hinson, MD, PhD,2 Lucia M. Blasucci, RN,1

Jennifer Zimmerman, RN,2 Wenqing Fan, MS,1

Tiffany Nguyen, MBA,3 and Ann Hsu, PhD3

1Department of Neurological Sciences, Rush UniversityMedical Center, Chicago, Illinois; 2Department of

Neurosciences, Medical University of South Carolina,Charleston, South Carolina; 3IMPAX Laboratories, Inc.,

Hayward, California

Video

Abstract: The objective is to test feasibility and utility ofhome-based videos for assessing Parkinson’s disease (PD)patients. As part of a clinical trial, patients opted betweencoming to the study sites or learning to videotape assess-ments at home. Those opting for at-home filming com-pleted training on videotape techniques. Ten-minute filmswere taken at 30-minute intervals over 8.5 hours, 2 and 4weeks after study entry using a protocol covering mostitems of the UDPRS motor examination and all Rush Dys-kinesia Rating Scale items. After each filming, patientsmarked their ON/OFF status, based on prior training.We determined the number of patients who elected self-taping and the quality of video segments obtained. Toassess ON/OFF patient accuracy, we compared the rater’sand patient’s assessment of ON/OFF at each time point.Of 12 participants, 10 elected self-videotaping and only 1time point was missed (99.5% taping compliance). Allself-recorded video segments were clear with all protocolelements included. With the exception of one missed ON/OFF rating, patient-based self-ratings occurred on time.Rating ON/OFF, UPDRS, and RDRS assessments for 8.5

This article includes supplementary material, available online athttp://www.interscience.wiley.com/jpages/0885-3185/suppmat.These data were obtained as part of an investigator-initiated study

supported by Impax Laboratories. CGG has served as a paid consultantfor Impax Laboratories and SW, TN, and AH are employees of ImpaxLaboratories. The authors have complied with all rules regarding thejournal’s ghost-writing policies.

*Correspondence to: Dr. Christopher G. Goetz, Rush UniversityMedical Center, Suite 755, 1725 W. Harrison Street, Chicago, IL60612, USA. E-mail: [email protected]

Received 31 January 2008; Revised 5 April 2008; Accepted 14April 2008

Published online 30 May 2008 in Wiley InterScience (www.


1479EVALUATING PARKINSON’S DISEASE PATIENTS AT HOME


hours required 170 minutes by the blinded rater. In spiteof patient training, mean ON/OFF concordance betweenrater and patients was only 64%. At home video-basedself-recordings are feasible and allow accurate rater-basedON/OFF assessments. In this group of patients with no ormild fluctuations, in spite of pretrial training, patientswere inaccurate in separating ON vs. OFF status. � 2008Movement Disorder Society

Key words: Parkinson’s disease; rating tools; randomizedclinical trials; ON/OFF

Parkinson’s disease (PD) assessment relies on rating

scales of Parkinsonism, dyskinesia, and motor fluctua-

tions (ON/OFF).1 For Parkinsonism, the most advocated

scale is Unified Parkinson’s Disease Rating Scale

(UPDRS) motor section.2 The Rush Dyskinesia Rating

Scale (RDRS) focuses on rater-based scores of dyskine-

sia impact on patient function during specified activ-

ities.3 Motor fluctuations are more difficult to assess

objectively because of long visits. Patient-based diaries

are utilized, although studies in other medical conditions

question patient compliance.4,5 The current feasibility

and utility study tested a new methodology whereby PD

patients participating in a clinical trial had the option to

come to the study sites for efficacy evaluations or to be

trained to record themselves at home.

METHODS

The study involved patients with PD6 with no or

mild motor fluctuations, (clinically pertinent to the

patient, but with no change in Hoehn and Yahr stage

between ON and OFF periods) and no troublesome

dyskinesias (scores of 0–1 on UPDRS items Part IV,

32–39), currently being treated with CD/LD 25/100 at

8 am, noon, 4 pm, and 8 pm. After signing informed

consent, all subjects were encouraged to be trained to

use a home video camera. If they were uncomfortable

with self-recording, they could return to the study cen-

ters at 2 and 4 weeks for 8.5 hours of testing. To qual-

ify for home filming, they needed to demonstrate suc-

cessful completion of the filming protocol. The time

for training was recorded. The filming protocol

involved 10-minute recordings every 30 minutes (8 am

to 4 pm) at 2 and 4 weeks after enrollment. The proto-

col followed the RDRS3 as well as tasks from the

UPDRS motor section and a self-assessment of ON/

OFF based on the 10 minutes of filming. This protocol

allowed a total score to be generated for the RDRS

based on Speech, Drinking, Dressing and Walking (total

score range 0–16) and a video-UPDRS score based on

Speech, Facial expression, Finger taps, Hand move-

ments, Leg agility, Arising from Chair, Posture, Gait,

Rest tremor and Body bradykinesia (total score range

0–68)2 [Appendix: Instructions and Video Recording

Script, Supplemental Materials]. For efficiency and

safety, Rapid alternating movements, Postural tremor,

and Postural stability were not included. Further, rigidity

could not be assessed on videotape. To ensure that these

deleted items did not significantly impact on the overall

assessment of Parkinsonism, we separately assessed 496

outpatients with PD and demonstrated that the modified

UPDRS correlated with the full UPDRS motor score

(Pearson correlation 0.979, P < 0.0001). Further, the

video UDPRS score correlated with the deleted items

(Pearson correlation 0.848, P < 0.001).

For ON/OFF education, subjects watched a video-

tape,7 followed by individualized counseling, and rated

themselves every 30 minutes over a 4-hour drug cycle

(ON without dyskinesia, ON with nontroublesome dys-

kinesias, ON with troublesome dyskinesias, and OFF).

Patients were instructed to consider dyskinesia only

during ON periods and OFF; dystonia was designated

as not part of dyskinesia rating. Eighty percent con-

cordance with the rater monitoring the patient’s status

was required for study participation.

The treatment protocol itself is the focus of a separate

report8; briefly, patients compared two formulations of

CD/LD in two 2-week crossover treatment phases. On

the last day of each treatment, patients were filmed every

30 minutes over 8.5 hours with the protocol as above

and patients then rated ON/OFF function covering the

filming time. Patients also completed 24-hour diaries on

the 2 days preceding the videotaping, and these docu-

ments were planned to be analyzed if patients retained

80% concordance with the blinded rater on the films.

At the end of the study, CGG, blinded to the treat-

ment order and the patient evaluations, rated each

10 minutes segment for UPDRS, RDRS, and ON/OFF.

For feasibility testing, we tabulated numbers of patients

who elected at-home vs. office filming. Among the

home-based tapes, we counted segments that followed

the protocol and provided clear data for full ratings.

We also measured concordance between the rater’s and

the patients’ ON/OFF ratings.

RESULTS

Thirteen consecutive patients who met inclusion cri-

teria were screened, offered participation, and signed

informed consent. All completed ON/OFF training in

one session (mean 4.34 hours, range 3.5–7.0). No sub-

ject had prior experience with video equipment. Eleven

1480 G. GOETZ ET AL.


subjects elected video training and all completed suc-

cessfully (mean 1.91 hours, range 0.5–8). All but one

needed a single training session, and this patient,

although successfully trained after two sessions (8 hours

total) withdrew from the program prior to drug expo-

sure because of continued concerns about his filming

competence. Ten patients, therefore, formed the at-

home self-video cohort. From the onset, two patients

elected to travel to their study center for assessments.

This report concerns the 12 patients who were exposed

to medication, six from each center and all completing

the two phases of the 4-week trial.

The 9 men and 3 women in the trial had a mean age

of 69 years, SD 11.0, and mean PD duration of 6 years,

SD 3.4. At study entry, the mean UPDRS ON motor

score was 20.5 (SD 5 6.7). An OFF motor score was

not performed but all were Hoehn and Yahr Stage II

ON and OFF. No patient had troublesome dyskinesias

by diary ratings or rater observation at baseline.

The 10 participants who filmed themselves were

compliant, with only 1/340 anticipated film segments

(10 patients 3 17 segments/phase 3 2 phases) missing

(99.7% taping compliance). All video segments were

clear with all protocol items included. There were no

equipment failures. For the two patients filmed by the

study staff, all video segments were complete and

clear. For ON/OFF compliance, the video protocol

included directions to complete the ON/OFF assess-

ment the end of each segment. All entries except the

time point error cited above were completed and on

time (99.7% compliance). With the modified UPDRS

motor assessment scored every half hour over the two

days or rating, the mean ON rating was 11.5 (SD 4.7)

and the mean OFF rating was 21.6 (5.6).

In spite of successful training and required concord-

ance standards to enter the study, the mean agreement

between rater and patient on ON/OFF was only 64%,

SD 5 19.4, range (29–85%) during the program. At

2 weeks, the concordance was 69% SD 5 20.9, range

(18–94%), and at 4 weeks, it dropped to 59% SD 527.3, range (12–88%). At 4 weeks, only four subjects

retained 80% or higher concordance with the rater.

Patients faltered on differentiating ON from OFF and

differentiating ON without dyskinesias from ON with

non-troublesome dyskinesias. Of 408 ON/OFF ratings,

56 discordant ON ratings occurred, 43 with the rater

identifying non-troublesome dyskinesia that was not

perceived by the patient and 13 where the patient iden-

tified dyskinesia when the rater saw none. There were

89 ratings discordant between ON without dyskinesia

and OFF ratings and an additional 18 times where

patients rated themselves as OFF, when the rater cate-

gorized them as ON with non-troublesome dyskinesia.

Because of the low concordance rates on the video-

based ON/OFF ratings, we did not analyze the 24 hour

unsupervised diaries.

DISCUSSION

Home-based self-filming was largely preferred by our

patients. The group was not selected based on prior ex-

perience with video recording, so we are confident that

we did not bias the sample with a technically sophisti-

cated group. Home-based video data were uniformly

clear and easy to rate. Patients were highly compliant,

allowing us to document both the timeliness of ON/OFF

assessments and to rate patient function objectively

without reliance on self-assessments. For small and me-

dium sized studies, we feel the at-home filming is practi-

cal and ensures very accurate information. Some patients

were uncomfortable filming themselves, and therefore

we recommend that patients be provided with options of

home- or office-based assessments. The high proportion

of patients favoring the at-home method encourages us

that this technique allows a new method to engage

patients for clinical research trials who otherwise might

decline for reasons of travel distance.

We retained the full RDRS but eliminated a few

items from the UDPRS. Based on the high correlations

between the video-based UPDRS and the full motor

UPDRS score as well as with the UPDRS scores from

the omitted items, we consider this scale to be an accu-

rate, though not comprehensive, reflection of patients’

overall Parkinsonism. For ON/OFF function, the se-

quential videotapes allowed the rater a rapid and easy

technique to score motor fluctuations. The total time to

review each patient over 8.5 hours was 170 minutes

or less of the blinded rater’s time. The comparable pro-

tocol in an office setting would have covered the full

8.5 hours, plus travel time for the patient and preparation

time for the staff, with the same rating time required.

We have not performed a cost-analysis of the difference,

but in our view, the modest cost of equipment (approxi-

mately $200 USA) and modest video training time out-

weighed the alternative of long office days.

We were impressed by the failure of patients to retain

their training knowledge of ON/OFF function and the re-

sultant inaccuracy of the ON/OFF self-assessments in

comparison to the rater. Admittedly, the judgment of

patient inaccuracy is anchored in the use of the rater’s

score as the assumed standard, and an alternative inter-

pretation is that the rater poorly determined ON/OFF

status; however, patients and enrolling investigators

agreed at study outset, and the standard for ON/OFF is

1481EVALUATING PARKINSON’S DISEASE PATIENTS AT HOME


traditionally based on the rater’s evaluation. Our study

population included only patients with no or mild fluctu-

ations and no troublesome dyskinesias, so that the dis-

tinction between ON and OFF or between ON without

and ON with non-troublesome dyskinesia was more

subtle than would be seen in patients with marked fluctu-

ations. Mild fluctuations may be difficult for patients to

assess on an on-going basis, and this observation under-

scores the value of this objective rating system, espe-

cially for these more subtle cases. A prior analysis of

teaching tapes documented retained patient knowledge

on ON/OFF function over one month of follow-up in

subjects with more marked fluctuations than those in the

current program.7 Reliance on home-based diaries with-

out rater verification remains problematic however even

in these cases of more marked fluctuation, because after

another video-based training program, 20% of patients

still did not understand the distinctions between ON and

OFF.9 We consider efforts to maximize ON/OFF assess-

ment accuracy extremely important, in light of the fre-

quent use of ON/OFF diary data as a pivotal outcome in

clinical trials.10–12 To test the ultimate utility of our new

methodology, we admittedly need to include patients

with all levels of fluctuations and with all levels of dys-

kinesias. Another problem considered by this methodol-

ogy is confirmation of patient compliance. Studies in

other medical conditions have documented very low

compliance with home-based diaries, with patients often

summarizing long-periods of time in recollection rather

than completing the assessments according to protocol

directions.4 In this protocol, because patients were

filmed and their diary completion was part of the filming

protocol, we could verify that patients completed their

diary rating exactly on time. As such, for small and me-

dium-sized studies, and especially in the context of mild

fluctuations, we advocate direct visual techniques as uti-

lized by this at-home filming methodology to ensure that

a trained observer makes the determination of functional

status. In our view, the widespread familiarity in the

United States with home entertainment electronic equip-

ment offers an opportunity for creative research proto-

cols that can minimize required study center visits with-

out reducing data quality and without relying on patient

assessments of motor function.

LEGENDS TO THE VIDEO

Demonstration of a patient performing the at-home

video-based protocol (see text and Appendix for link-

age between specific tasks and ratings on the UPDRS,

RDRS). A final assessment by the patient assesses

motor fluctuation status, but the patient does not show

this rating so that the trained rater is not influenced by

the patient’s self-assessment. Text of the video proto-

col is available as Supplemental Material Appendix

available on the journal website.

REFERENCES

1. Cubo E, Goetz CG. Methods of patient assessment in surgicaltherapy for movement disorders. In: Tarsy D, Vitek JL, LozanoAM, editors. Surgical treatment of Parkinson’s disease and othermovement disorders. Totowa, NJ: Humana Press; 2003. p 69–85.

2. Fahn S, Elton RL, UPDRS program members. Unified Parkinson’sDisease Rating Scale. In: Fahn S, Marsden CD, Goldstein M,Calne DB, editors. Recent developments in Parkinson’s disease.Florham Park, NJ: Macmillan Healthcare Information; 1987.Vol. 2, pp 153–163 and pp 293–304.

3. Goetz CG, Stebbins GT, Shale HM, et al. Utility of an objectivedyskinesia rating scale for Parkinson’s disease inter- and intra-rater reliability assessment. Mov Disord 1994;9:390–394.

4. Hauser RA, Deckers F, Lehert P. Parkinson’s disease home diary:further validation and implications for clinical trials. Mov Disord2004;19:1409–1413.

5. Stone AA, Shiffman S, Schwartz JE, et al. Patient compliancewith paper and electronic diaries. Control Clin Trials 2003;24:182–199.

6. CAPIT Committee: Langston JW, Widner H, Goetz CG, et al.Core assessment program for intracerebral transplantations(CAPIT). Mov Disord 1992;7:2–13.

7. Goetz CG, Stebbins GT, Blasucci LM, Grobman M. Efficacy ofa patient-training videotape on motor fluctuations for ‘‘on-off’’diaries in Parkinson’s disease. Mov Disord 1997;12:1039–1041.

8. Goetz CG, Leurgans S, Hinson VK, et al. New formulation ofcarbidopa/ levodopa (IPX504, VADOVA IR1ER) vs. standardcarbidopa/levodopa in stable PD patients Mov Disord 2007;22(Suppl 16):S244.

9. Hauser RA, Russ H, Haeger DA, Brugiere-Fontenille M, MullerT, Wenning GK. Patient evaluation of a home diary to assess du-ration and severity of dyskinesia in Parkinson’s disease. MovDisord 2006;29:322–230.

10. Adler CH, Singer C, O’Brien C. Randomized, placebo-controlledstudy of tolcapone in patients with fluctuating Parkinson’s diseasetreated with levodopa-carbidopa. Arch Neurol 1998;55:1089–1095.

11. Parkinson Study Group. Entacapone improves motor fluctuationsin levodopa-treated Parkinson’s disease patients. Ann Neurol1997;42:747–755.

12. Pinter MM, Pogarell O, Oertel WH. Efficacy, safety and toler-ance of the non-ergoline dopamine agonist pramipexole in thetreatment of advanced Parkinson’s disease: a double-blind pla-cebo controlled randomized multicentre study. J Neurol Neuro-surg Psychiatry 1999;66:436–441.

1482 G. GOETZ ET AL.


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