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Brief Reports

LRRK2 Variation and Parkinson’sDisease in African Americans

Owen A. Ross, PhD,1* Greggory J. Wilhoite, BS,1

Justin A. Bacon, BS,1 Alexandra Soto-Ortolaza, BS,1

Jennifer Kachergus, BS,1 Stephanie A. Cobb, BA,1

Andreas Puschmann, MD,1 Carles Vilarino-Guell, PhD,1

Matthew J. Farrer, PhD,1 Neill Graff-Radford, MD,2

James F. Meschia, MD,2 and Zbigniew K. Wszolek, MD2

1Department of Neuroscience, Mayo Clinic College ofMedicine, Jacksonville, Florida, USA; 2Department of

Neurology, Mayo Clinic College of Medicine,Jacksonville, Florida, USA

Abstract: The global impact of LRRK2 mutations is yet tobe realized with a lack of studies in specific ethnic groups,including those of Asian and African descent. Herein, weinvestigated the frequency of common LRRK2 variants bycomplete exon sequencing in a series of publicly availableAfrican American Parkinson’s disease patients. Our studyidentified three novel synonymous exonic variants and 13known coding variations; however, there did not appearto be any frequent (>5%) pathogenic mutations. Giventhe ethnic-specific LRRK2 variation previously identifiedin PD further studies in under-represented populationsare warranted. � 2010 Movement Disorder Society

Key words: parkinsonism; leucine-rich repeat kinase 2;genetics

Leucine-rich repeat kinase 2 (LRRK2) mutations are

recognized as the most frequent genetic cause of Par-

kinson’s disease (PD) identified to date. Although only

a small number of the variations within the gene are

known to affect disease risk (n 5 8), the pathogenicity

of many others remains equivocal. LRRK2 mutations

highlight many of the phenomena associated with late-

onset sporadic PD including pleomorphism in pheno-

typic presentation and a large variation in the disease

penetrance. This has been exemplified by carriers of

Lrrk2 p.R1441C and p.G2019S with a diverse range in

age-at-onset of PD.1,2 Another prominent feature of

LRRK2 parkinsonism is the relatively high frequency

of specific mutations observed in ethnically distinct

populations.3–6

Mutations in LRRK2 are distributed across the gene

and affect different domains of the protein, which

makes screening an expensive and time-consuming

proposal. For this reason, the great majority of samples

sequenced for the full 51 exon LRRK2 gene have been

Caucasian samples from Western Europe or North

America. Only a relatively small number of samples

have been sequenced in African or Asian populations,

although the latter have been fruitful identifying two

common risk factors (Lrrk2 p.R1628P and

p.G2385R).3,6,7 Genetic studies of PD in sub-Saharan

Africa are rare8–11; however, a number of studies have

recently been published regarding the observed high

frequency of Lrrk2 p.G2019S (30–40% of patients) in

the Maghreb region of North Africa.2,4,12,13

To assess the full impact of LRRK2 mutations on the

global PD community, it is crucial that full gene

sequencing is performed on different ethnic groups. In

this study, we set out to sequence subjects of African

American declared ethnicity with PD to assess the fre-

quency of common LRRK2 variation in this population.

SUBJECTS AND METHODS

DNA samples from African American patients with

PD were obtained from the Coriell Cell Repository

collection (Coriell Institute for Medical Research,

Camden, NJ) under the appropriate ethical approval.

Our series contained 22 patients (12 female and 10

male) with an average age-at-onset of 50.6 years

(range 27–65 years) and an average age-at-study of

58.7 years (range 40–72 years). A family history of

parkinsonism was reported in 6 patients (27%).

Unfortunately given the nature of samples (Coriell Cell

Repository), the clinical records are limited. To estab-

lish allele frequencies, we screened 92 African

*Correspondence to: Dr. Owen A. Ross, Molecular Genetics Labo-ratory and Core, Department of Neuroscience, Morris K. Udall Par-kinson’s Disease Research Center of Excellence, Mayo Clinic, 4500San Pablo Road, Jacksonville, Florida 32224.E-mail: [email protected]

Potential conflict of interest: None.Received 13 October 2009; Revised 19 February 2010; Accepted

17 March 2010Published online 28 July 2010 in Wiley Online Library

(wileyonlinelibrary.com). DOI: 10.1002/mds.23163

1973

Movement DisordersVol. 25, No. 12, 2010, pp. 1973–1992� 2010 Movement Disorder Society

American control subjects (67 female and 25 male)

obtained through the Coriell Repository or collected at

Mayo Clinic Florida with an average age-at-study of

41.9 years (range 20–71 years). In addition, a series of

420 US Caucasians controls collected at Mayo Clinic

Florida (196 female and 224 male) were genotyped to

provide allelic frequencies; this series had an average

age-at-study of 73.4 years (range 34–93 years).

Primers were designed for sequencing of the entire

coding region of the LRRK2 gene (51 exons) and

exon-intron boundaries (Available on request). PCR

products were generated on 384 well plates using

standard protocols. A Biomek FX robot allows highly

efficient, easily automated magnetic-bead based PCR

purification system (AMPure beads, Agencourt, Bev-

erly, MA) that requires no sample transfer, centrifuga-

tion, or filtration steps. The same bead technology

(CleanSEQ beads, Agencourt) allows for sequencing

reactions to be performed in 10 lL volumes containing

5lL of clean PCR product, 1:32 dilution of BigDye

Terminator and 3.2 pmol of primer. After the removal

of dye-terminator using the Biomek FX robot and mag-

netic-bead technology, sequencing products are electro-

phorized on an ABI 3730 capillary array and the data

analyzed with SeqScape software (ABI).

RESULTS

We identified 16 exonic variants in the 22 African

American PD patients, three novel synonymous

changes (p.A75A, p.V674V, and p.S1721S) were

observed and are shaded in Table 1. The three com-

mon variants highlighted with an asterisk represent a

switch of the minor and major allele designations from

that observed in Caucasian samples. Control frequen-

cies are provided for a series of 92 African American

and 420 Caucasian US subjects. Linkage disequili-

brium measures are provided as r2 values within the

African American subjects (Fig. 1).14 In addition, there

was no evidence of any intron-exon boundary variants

that would result in alternate splicing.

DISCUSSION

Ongoing studies by our group and others are

attempting to resolve the pathogenicity, prevalence and

penetrance of LRRK2 coding variants in PD. There is a

gap in our knowledge regarding the frequency and

impact of LRRK2 mutations on PD in distinct ethnic

groups of people. To this goal, we performed sequenc-

ing of 22 African American PD patients for the entire

LRRK2 gene identifying a number of known and novel

exonic variations (Table 1). There was neither evi-

dence of any common (>5%) pathogenic mutations

nor of known variants of high (e.g., p.G2019S) or low

penetrance (e.g., p.R1628P) in these samples from

African American PD patients.2,6

This study does not rule out a role for LRRK2 muta-

tions in PD patients of African descent but does show

that unlike some populations from North Africa there

TABLE 1. Observed 16 exonic variants in the LRRK2 sequencing of 22 African American PD patients

Exon SNP c.LRRK2 p.Lrrk2Carriers(n 5 22)

MAF in AAPD (n 5 44)

MAF in AAcontrols (n 5 184)

MAF in CaucasianUS controls (n 5 840)

Exon 1 rs2256408 149A>G H50R 5 0.11 0.09 0.001Exon 2 rs75054132 225G>A A75A 1 0.02 0.01 0.00Exon 5 rs10878245 457T>C L153L 17 0.39 0.43 0.64Exon 14 rs7308720 1653C>G N551K 9 0.20 0.12 0.07Exon 17 rs72546319 2022A>C V674V 1 0.02 0.00 0.00Exon 22 rs7966550 2857T>C L953L 1 0.02 0.02 0.12Exon 30 rs7133914 4193G>A R1398H 6 0.14 0.12 0.07Exon 30 rs11175964 4269G>A K1423K 2 0.05 0.03 0.06Exon 34 ars1427263 4872A>C G1624G 9 0.20 0.24 0.33Exon 34 rs11176013 4911A>G K1637K 21 0.48 0.59 0.57Exon 34 rs11564148 4939T>A S1647T 7 0.16 0.19 0.31Exon 35 rs79909111 5163A>G S1721S 1 0.02 0.00 0.004Exon 37 rs10878371 5457T>C G1819G 20 0.45 0.61 0.43Exon 43 rs10878405 6324G>A E2108E 6 0.14 0.16 0.30Exon 48 rs33962975 7155A>G G2385G 4 0.09 0.05 0.14Exon 49 ars3761863 7190C>T T2397M 18 0.41 0.46 0.34

Novel synonymous variants identified in this study are shaded.The MAF for each variant is shown for the 22 African American patients, 92 African American controls, and 420 Caucasian controls (all sub-

jects are US citizens).MAF, minor allele frequency; AA, African American.aThe minor and major allele designations have switched from that normally observed in Caucasians.

1974 O.A. ROSS ET AL.

Movement Disorders, Vol. 25, No. 12, 2010

is no highly prevalent pathogenic mutation. Our sample

size although relatively small (n 5 22) reflects the

comprehensive sequencing approach of the all 51

exons of the LRRK2 gene and represents a large body

of data. However, this study does not account for pos-

sible variation in noncoding regions of the gene which

may account for disease risk. Gene sequencing studies

are important as we examine different ethnic groups

for LRRK2 variants given the knowledge of the ethnic

frequency differences that already exists.

These results represent our preliminary studies to

characterize the frequency of LRRK2 variants in the

African American population. Differences are observed

for the minor allele frequencies of a number of LRRK2variants between African American and Caucasian sub-

jects which will be an important consideration for

future association studies (Table 1). To date, over 75

nonsynonymous substitutions have been reported with

the pathogenicity and frequency of these yet to be

established in a number of different populations. The

novel variants identified in this study in these African

American patients will be included in our future

LRRK2 genetic studies.

Acknowledgments: Mayo Clinic Jacksonville is a MorrisK. Udall Parkinson’s Disease Research Center of Excellence(NINDS P50 #NS40256). MJF and OAR are supported by a

grant from the Michael J. Fox foundation. ZKW is also par-tially funded by NIH P01 AG017216, R01 NS057567, R01AG015866, CIHR 121849, and PARF C06-01. This studyused samples from the NINDS Human Genetics ResourceCenter DNA and Cell Line Repository (http://ccr.coriell.org/ninds), as well as clinical data. NINDS Repository samplenumbers corresponding to the samples used are: ND04381;ND05017; ND00165; ND20308; ND01131; ND01078;ND02892; ND00079; ND05171; ND14044; ND06940;ND01137; ND05602; ND06487; ND14506; ND14907;ND01985; ND14630; ND05460; ND06105; ND01177;ND04160. We would like to thank all those who have contrib-uted to our research, particularly Jill A Searcy for assistance.

Financial Disclosures: Owen A. Ross: Employment:Mayo Clinic; Grants: Michael J. Fox Foundation and Ameri-can Heart Association. Greggory J. Wilhoite, Justin A. Bacon,Alexandra Soto-Ortolaza, Jennifer Kachergus, Stephanie A.Cobb, Andreas Puschmann, Carles Vilarino-Guell: Employ-ment: Mayo Clinic. Matthew J. Farrer: Dr. Farrer reports a USprovisional patent application for a device that treats neurode-generative diseases, which has been licensed to Alnylam Phar-maceuticals Inc. Dr Farrer also reports an honorarium for aseminar from H. Lundbeck A/S, GlaxoSmithKline, Elan Phar-maceuticals and Genzyme; Advisory Boards: Michael J. FoxFoundation; Employment: Mayo Clinic; Grants: NIH and Mi-chael J. Fox Foundation. James F. Meschia: Employment:Mayo Clinic; Grants: NINDS and the Hanley Foundation(DC). Neill Graff-Radford: Employment: Mayo Clinic; Scien-tific Advisory Board: Codman. Zbigniew K. Wszolek:Employment: Mayo Clinic; Grants: NIH and PARF.

FIG. 1. The linkage disequilibrium between the 16 LRRK2 variants observed within the African American samples is highlighted in a Haploviewplot with r2 values shown and represented in shading.14 All African American patients and controls were used to generate the plot.

1975LRRK2-PARKINSONISM IN AFRICAN AMERICANS


Author Roles: Owen A. Ross was involved in funding,conception, and organization of research project; drafting,review and critique of manuscript. Greggory J. Wilhoite, Jus-tin A. Bacon, Alexandra I. Soto-Ortolaza, Jennifer Kacher-gus, Stephanie A. Cobb, and Andreas Puschmann wereinvolved in execution of research project; review and critiqueof manuscript. Carles Vilarino-Guell was involved in concep-tion and organization of research project, review and critiqueof manuscript. Matthew J. Farrer was involved in fundingand conception of research project, review and critique ofmanuscript. Neill Graff-Radford was involved in funding,sample collection, review and critique of manuscript. JamesF. Meschia was involved in funding, sample collection,review and critique of manuscript. Zbigniew K. Wszolek wasinvolved in funding, conception, and organization of researchproject; sample collection; review and critique of manuscript.

REFERENCES

1. Haugarvoll K, Rademakers R, Kachergus JM, et al. Lrrk2R1441C parkinsonism is clinically similar to sporadic Parkinsondisease. Neurology 2008;70(16 Part 2):1456–1460.

2. Hulihan MM, Ishihara-Paul L, Kachergus J, et al. LRRK2Gly2019Ser penetrance in Arab-Berber patients from Tunisia: acase-control genetic study. Lancet Neurol 2008;7:591–594.

3. Di Fonzo A, Wu-Chou YH, Lu CS, et al. A common missensevariant in the LRRK2 gene, Gly2385Arg, associated with Parkin-son’s disease risk in Taiwan. Neurogenetics 2006;7:133–138.

4. Lesage S, Durr A, Tazir M, et al. LRRK2 G2019S as a cause ofParkinson’s disease in North African Arabs. N Engl J Med 2006;354:422–423.

5. Ozelius LJ, Senthil G, Saunders-Pullman R, et al. LRRK2G2019S as a cause of Parkinson’s disease in Ashkenazi Jews. NEngl J Med 2006;354:424–425.

6. Ross OA, Wu YR, Lee MC, et al. Analysis of Lrrk2 R1628Pas a risk factor for Parkinson’s disease. Ann Neurol 2008;64:88–92.

7. Mata IF, Kachergus JM, Taylor JP, et al. Lrrk2 pathogenic sub-stitutions in Parkinson’s disease. Neurogenetics 2005;6:171–177.

8. Atadzhanov M, Zumla A, Mwaba P. Study of familial Parkin-son’s disease in Russia, Uzbekistan, and Zambia. Postgrad Med J2005;81:117–121.

9. Okubadejo N, Britton A, Crews C, et al. Analysis of Nigerianswith apparently sporadic Parkinson disease for mutations inLRRK2, PRKN and ATXN3. PLoS ONE 2008;3:e3421.

10. Okubadejo NU. An analysis of genetic studies of Parkinson’s dis-ease in Africa. Parkinsonism Relat Disord 2008;14:177–182.

11. Okubadejo NU, Bower JH, Rocca WA, Maraganore DM. Parkin-son’s disease in Africa: a systematic review of epidemiologicand genetic studies. Mov Disord 2006;21:2150–2156.

12. Ishihara L, Gibson RA, Warren L, et al. Screening for Lrrk2G2019S and clinical comparison of Tunisian and North Ameri-can Caucasian Parkinson’s disease families. Mov Disord 2007;22:55–61.

13. Ishihara L, Warren L, Gibson R, et al. Clinical features of Par-kinson disease patients with homozygous leucine-rich repeat ki-nase 2 G2019S mutations. Arch Neurol 2006;63:1250–1254.

14. Barrett JC. Haploview: visualization and analysis of SNP geno-type data. CSH Protoc 2009;2009(10):pdb ip71.

Impaired Insulin Sensitivity andSecretion in NormoglycemicPatients with Spinocerebellar

Ataxia Type 1

Nebojsa M. Lalic, MD, PhD,1

NatasA Dragasevic, MD, PhD,2

Elka Stefanova, MD, PhD,2

Aleksandra Jotic, MD, PhD,1

Katarina Lalic, MD, PhD,1 Tanja Milicic, MD,1

Igor Petrovic, MD,2 Marija Macesic, MD,1

and Vladimir S. Kostic, MD, PhD2*

1Clinical Center of Serbia, Institute for Endocrinology,Diabetes and Metabolic Diseases, Belgrade, Serbia;2Clinical Center of Serbia, Institute of Neurology,

Belgrade, Serbia

Abstract: We have recently shown an impairment in insu-lin sensitivity and insulin secretion in normoglycemicpatients with Huntington disease (HD). To investigatewhether such observations are HD-specific or may becommon to other polyglutamine diseases, glucose homeo-stasis was studied in 12 unrelated, untreated normogly-cemic patients with spinocerebellar ataxia type 1 (SCA1),another entity from the family of polyglutamine diseases,and 24 healthy, matched controls. Metabolic investiga-tions included (a) glucose tolerance assessment on the ba-sis of glucose curve during oral glucose challenge; (b) in-sulin sensitivity assessment by the homeostasis modelassessment (HOMA) and the euglycemic insulin clamp (Mvalue); and (c) insulin secretion by acute insulin response(AIR) and insulinogenic index. The evaluation of insulinsensitivity demonstrated higher HOMA-insulin resistanceindices, and lower M values (P < 0.001 and P < 0.05,respectively), while both the AIR and the insulinogenicindex were lower in patients with SCA1 compared to con-trols (P < 0.001 and P < 0.05, respectively). Our datasuggested an impairment in insulin secretion capacity, aswell as simultaneous decrease in insulin sensitivity, withan increase in insulin resistance level in patients withSCA1. � 2010 Movement Disorder Society

Key words: spinocerebellar ataxia; insulin resistance

State on ghost-writing: There is no ghost-writing by any one notnamed on the author list

*Correspondence to: Vladimir S. Kostic, Institute of NeurologyCCS, Ul. Dr Subotica 6, Belgrade 11000, Serbia.E-mail: [email protected].

Potential conflict of interest: Nothing to report.Received 4 December 2009; Revised 2 February 2010; Accepted



1976 N.M. LALIC ET AL.


The disease-causing mutation of the autosomal dom-

inant spinocerebellar ataxia type 1 (SCA1) is the

expansion of an unstable CAG trinucleotide repeat

(normal range: 6–44; range in affected alleles: 39–91)

within the coding region of the gene on the short arm

of chromosome 6, producing an expanded tract of

glutamine amino acids within the SCA1 product,

ataxin-1.1

An increasing number of neurodegenerative disor-

ders are frequently associated with diabetes mellitus

(DM) or its antecessors, insulin resistance and/or

impaired glucose tolerance.2 Hypothetically, these find-

ings may be nonspecific and can be attributed to the

chronic illness rather than to neurodegenerative disease

per se. We have recently shown an impairment in insu-

lin sensitivity and insulin secretion in normoglycemic

patients with HD.3 To investigate whether such obser-

vations are HD-specific or may be common to other

polyglutamine (polyQ) diseases, we studied possible

changes in insulin sensitivity and secretion in a group

of consecutive normoglycemic patients with SCA1.

PATIENTS AND METHODS

After providing informed consent, 12 consecutive

unrelated nondiabetic patients (according to the WHO

criteria for the diagnosis of DM)4 with genetically veri-

fied SCA1, who were not bed- or wheelchair-bound

[Activities of Daily Living (ADL) scores5 between 60

and 90 in all the patients], were recruited from 12 dif-

ferent families identified as previously described.6

Except for physical therapy and vitamins, none of

them received any treatment that may influence insulin

homeostasis (ie, neuroleptics). The control group com-

prised 24 healthy subjects matched by age (62 years),

sex, and socioeconomic background (Table 1). The

study was approved by the Ethics Committee of the

Clinical Center of Serbia.

Assessments of the body mass index (BMI), body

fat, and fat-free mass (bioimpedance balance), waist

circumference and glucose homeostasis were done as

previously described3 and included: (1) glucose toler-

ance assessment based on the blood glucose curve dur-

ing the oral glucose tolerance test (OGTT); (2) insulin

sensitivity assessment using two different methods: (i)

homeostasis model assessment (HOMA) based on basal

plasma glucose (PG), and plasma insulin (PI) levels,

primarily evaluating hepatic insulin sensitivity, and (ii)

the euglycaemic insulin clamp determining predomi-

nantly peripheral insulin sensitivity; and (3) estimation

of the early phase insulin secretion using two different

methods: (i) the acute insulin response (AIR), deter-

mined during the intravenous glucose tolerance test

(IVGTT), and (ii) the insulinogenic index, determined

during the OGTT.

Shortly, the HOMA-insulin resistance (HOMA-IR)

index, describing insulin resistance as a reciprocal

value of insulin sensitivity, was calculated from the

following equation: (PG x PI)/22.5.3

The IVGTT was performed by infusing 0.3 g/kg

body weight of 50% glucose in 30 seconds and the

AIR was determined as the average increment of PI

higher than basal values for samples obtained in the

first 10 minutes of the IVGTT.

The insulinogenic index was determined during the

OGTT as the ratio of the increment of PI to that of PG

determined 30 minutes after a glucose load (DPI (0–30minutes)/DPG (0–30 minutes).

Euglycemic insulin clamp was performed by using

insulin (40 mU/min21/m22) and glucose infusion

simultaneously to achieve and maintain steady state at

target PG values at 5.0 mmol/L (615%). The steady-

state period was between 80 and 120 minutes. Insulin

sensitivity was expressed as a M value averaged over

the final 40 minutes of the two-hour clamp.7

The results were evaluated using a statistical soft-

ware program SPSS 10.0 (SPSS, Chicago, Illinois).

Comparisons of the mean values between groups were

performed using the unpaired t test for continuous vari-ables or the Mann-Whitney test for the insulinogenic

index as a variable found to be without normal distri-

bution. Spearman correlations were calculated to deter-

mine the relationship between the variables.

TABLE 1. Characteristics of patients with SCA1 andhealthy controls

SCA 1 Controls

Number of patients 12 24Gender (male/female) 7/5 13/11Age (yr) 37.7 6 1.66 36.9 6 1.16Disease duration (yr) 5.37 6 0.79 –BMI (kg/m2) 22.85 6 1.17 22.95 6 0.64Fat mass (kg) 17.09 6 2.06 15.59 6 1.26Fat-free mass (kg) 50.55 6 3.32 55.62 6 2.77Waist circumference (cm) 86.08 6 3.02 82.87 6 2.40FPG (mmol/L) 4.77 6 0.16 4.85 6 0.11Total cholesterol (mmol/L) 5.62 6 0.34a 4.69 6 0.16LDL cholesterol (mmol/L) 3.69 6 0.31a 2.91 6 0.15HDL cholesterol (mmol/L) 1.19 6 0.04 1.28 6 0.07Triglycerides (mmol/L) 1.39 6 0.16 1.04 6 0.17Insulin (mU/L) 10.55 6 0.89b 5.65 6 1.12

aP < 0.05.bP < 0.01 vs controls.BMI, body mass index; FPG, fasting plasma glucose levels; LDL,

low-density lipoprotein; HDL, high-density lipoprotein. Data areexpressed as means 6 SEMs.


1977INSULIN SENSITIVITY AND SECRETION IN SCA1

RESULTS

Insulin levels (P < 0.001), as well as total (P < 0.05)

and LDL cholesterol levels (P < 0.05), were signifi-

cantly higher in patients with SCA1 compared to con-

trols (Table 1). The analysis of the PG curves (not pre-

sented) during the OGTT has demonstrated that in both

groups they stayed within nondiabetic range. None of

the patients was excluded from the study because of the

results of OGTT or the presence of DM.

Evaluation of insulin sensitivity demonstrated that the

mean (6SEM) HOMA-IR index was significantly

higher in patients with SCA1 (2.61 6 0.21) compared to

controls (1.19 6 0.11, P < 0.001) (Fig. 1a). Further

analysis of insulin sensitivity using M value, being a

measure of total insulin-induced glucose uptake,

revealed significantly lower M value in patients with

SCA1 (6.71 6 0.74 mg/kgbw/min) compared to controls

(10.02 6 0.84 mg/kgbw/min; P < 0.05) (Fig. 1b).

The mean (6SEM) AIR values were significantly

lower in patients with SCA1 compared to controls

(30.68 6 7.53 vs 74.67 6 6.87 mU/L; P < 0.001)

(Figure 1c). Values of the insulinogenic index were

also lower in patients with SCA1 than in controls (5.11

6 1.54 vs 8.95 6 1.00; P < 0.05) (Figure 1d).

These differences remained significant after adjust-

ments for BMI and waist circumference.

In patients with SCA1 no significant correlations

were found between insulin sensitivity parameters (Mvalue, HOMA-IR) and anthropometric features (BMI,

fat mass and waist) (M value with BMI [r 5 20.140,

P 5 0.665], fat mass [r 5 20.259, P 5 0.417], and

waist [r 5 20.382, P 5 0.221], and HOMA-IR with

BMI [r 5 0.224, P 5 0.484], fat mass [r 5 0.084,

P 5 0.795] and waist [r 5 0.420, P 5 0.174]), while

in controls some of these correlations were detected

(M value with BMI [r 5 20.537, P < 0.05], fat mass

[r 5 20.611, P < 0.01], and waist [r 5 20.461, P <0.05], and HOMA-IR with BMI [r 5 0.414, P < 0.05]

and fat mass [r 5 0.431, P < 0.05]).

The mean number of CAG repeats (CAGn) in

patients with SCA1 was 52.8 (range, 44–64). No corre-

lation was observed between the CAGn and the

HOMA-IR index, the AIR, the sensitivity index and

the insulinogenic index, as well as between the impair-

FIG. 1. Results of the evaluation of insulin sensitivity using (a) the homeostasis model assessment–insulin resistance (HOMA-IR) index and (b)M value, and of the early phase insulin secretion using (c) the acute insulin response (AIR), and (d) the insulinogenic index in patients withSCA1 and controls. Values are given as means (6 SEMs). *P < 0.001 vs controls; **P < 0.05 vs controls.

1978 N.M. LALIC ET AL.


ments in glucose homeostasis and the onset or duration

of the disease.

DISCUSSION

The main finding is the impairment in the insulin

sensitivity in normoglycemic patients with SCA1, both

on the level of hepatic and peripheral tissues. Analysis

of early phase insulin secretion also demonstrated the

decrease in insulin secretion capacity after intravenous

and after a more potent oral glucose challenge that

involved the cephalic phase of hormone release (Fig-

ures 1). The lack of correlation between any of the

described parameters and the CAGn suggested (a) that

they might not be affected by the polymorphism of the

mutated SCA1 gene, (b) that this may be also due to a

limited range of expansions observed in this study

(within the first half of the range of expanded alleles),

or (c) a limited number of included patients.1

These data are similar, although not the same as

those we obtained in another polyQ disease, HD.3

Some of the differences between SCA1 and HD

related to cholesterol and triglyceride levels, as well

as a different trend of changes of BMI, may be a

consequence of higher energy expenditure caused by

involuntary movements in HD in comparison to

patients with SCA1, characterized with a more seden-

tary life style. However, except for some anecdotal

reports on increased glycemia levels in patients with

SCA1,8 we have no clinical studies focused on glu-

cose homeostasis in SCA1. In polyQ diseases some

of the abnormalities in peripheral tissues are not sec-

ondary to brain dysfunction, but most seem to be

directly caused by expression of the mutant protein in

peripheral tissues.9 The histopathologic hallmark of

HD, intranuclear inclusion, is also present in pancre-

atic cells.10 The accumulation of such insoluble nu-

clear protein aggregates in islets was temporally asso-

ciated with selective impairment of expression of

transcriptional regulatory proteins essential for glu-

cose-responsive insulin gene expression.11 Both wild

and mutant ataxin-1 were also expressed in non-neu-

ronal tissues, but specific data for pancreatic b-cellsin patients with SCA1 are lacking.12

Pathogenic and clinical significance of observed

impairments in glucose homeostasis in patients with

SCA1, if exists at all, is highly speculative. Recent

data point to the insulin/insulin-like growth factor 1

(IGF1) signaling (IIS) pathway as a major candidate to

link proteotoxicity (including polyQ aggregates),

aging, and late-onset neurodegenerative diseases.2

Cohen and Dillin2 suggested that each organism has

an optimal level of IIS and that both higher or lower

than optimal levels of IIS interfere with the metabo-

lism and proteotoxic mechanisms, with insulin resist-

ance seeming to be analogous to reduced insulin sig-

naling. One of the major IIS pathways mediating sur-

vival signals in neuronal cells, phosphatidylinositol-3-

kinase/Akt signaling, also acts through phosphorylation

of ataxin-1, promotiong its binding to 14–3-3 protein,

which in turn leads to ataxin-1 accumulation and neu-

rodegeneration.13 Insulin and IGF-I modulate macroau-

tophagy, which has been shown to participate in

the degradation and clearance of polyQ-containing

proteins.2

Among the limitations of our study is the small

number of SCA1 patients. Even among healthy per-

sons, measurements of insulin resistance may signifi-

cantly vary due to differences of weight, physical fit-

ness, and potential inflammatory processes, or reduced

motor activities of our patients due to ataxia. However,

we found that in the SCA1 patients, in contrast to the

healthy controls, there was no correlation between in-

sulin resistance and weight parameters including fat

mass and waist. Thus, we believe that our results are

still valuable as they are based on relatively homoge-

nous group of rare, unrelated patients according to age,

disease duration, BMI, waist circumference, and ADL

values (Table 1).

The present phenomenologic data of the simultane-

ous presence of insulin resistance and decreased early

phase b-cell secretion in normoglycemic patients with

SCA1 do not imply any causative or modifying

effect. Further longitudinal studies are necessary to

elucidate (1) the progression of observed impairments,

and (2) whether the modulation of insulin sensitivity

may also influence the course and expression of the

disease.

Acknowledgments: This work was funded by the Ministryof Science, Republic of Serbia (projects no. 145057 and145089).

Financial Disclosures: Natasa Dragasevic, Elka Stefa-nova, Tanja Milicic, Igor Petrovic, Marija Macesic: none;Nebojsa M. Lalic received Honoraria for lectures for SanofiAventis, Novo Nordisk, Servier, Merck Serono, and MSD;Aleksandra Jotic received Honoraria for lectures for NovoNordisk, Servier and Merck Serono; Katarina Lalic receivedHonoraria for lectures for Novo Nordisk, Servier and MerckSerono; Vladimir S. Kostic, member of Regional South-Eastern European Advisory Board of Boehringer Ingelheim,received Honoraria for lectures for Novartis, BoehringerIngelheim, Libra (Merck), Lundbeck and Glaxo-Smith-Kline.

Author Roles: Nebojsa M. Lalic was involved in concep-tion and organization of research project, design, execution,

1979INSULIN SENSITIVITY AND SECRETION IN SCA1


and review and critique of statistical analysis, writing of thefirst draft, and review and critique of manuscript; NatasaDragasevic was involved in organization and executionresearch project, and review and critique of manuscript;Elka Stefanova was involved in organization of researchproject, review and critique of statistical analysis, andreview and critique of manuscript; Aleksandra Jotic wasinvolved in organization and execution of research project,execution of statistical analysis, writing of the first draft ofmanuscript; Katarina Lalic was involved in organization andexecution of research project, execution of statistical analy-sis; writing of the first draft of manuscript; Tanja Milicicwas involved in organization and execution of research pro-ject and execution of statistical analysis; Igor Petrovic wasinvolved in organization and execution of research projectand execution of statistical analysis; Marija Macesic wasinvolved in organization and execution of research projectand execution of statistical analysis; Vladimir S. Kostic wasinvolved in conception and organization of research project,design, execution, and review and critique of statisticalanalysis, writing of the first draft and review and critique ofmanuscript.

REFERENCES

1. Orr HT, Zoghbi HY. Trinucleotide repeat disorders. Annu RevNeurosci 2007;30:575–621.

2. Cohen E, Dillin A. The insulin paradox: proteotoxicity and neu-rodegeneration. Nat Rev Neurosci 2008;9:759–767.

3. Lalic NM, Maric J, Svetel M, et al. Glucose homeostasis in Hun-tington disease. Abnormalities in insulin sensitivity and early-phase insulin secretion. Arch Neurol 2008;65:476–480.

4. World Health Organization, Definition and diagnosis of diabetesmellitus and intermediate hyperglycemia: report of a WHO/IDFconsultation. Geneva: World Health Org; 2006.

5. Schwab RS, England AC. Parkinsonism due to various specificcauses. In: Vinken PJ, Bruyn GW, editors. Handbook of clini-cal neurology, Vol. 6. Amsterdam, North Holland: 1968. p203–233.

6. Dragasevic N, Culjkovic B, Klein C, et al. Frequency analysisand clinical characterization of different types of spinocerebellarataxia in Serbian patients. Mov Disord 2006;21:187–191.

7. Ferrannini E, Mari A. How to measure insulin sensitivity.J Hypertens 1998;16:895–906.

8. Uchihara T, Takeda Y, Kobayashi T, et al. Unexpected clinico-pathological phenotype linked to small elongation of CAG repeatin SCA1 gene. J Neurol 2006;253:396–398.

9. van der Burg JMM, Bjorqvist M, Brundin P. Beyond the brain:widespread pathology in Huntington’s disease. Lancet Neurol2009;8:765–774.

10. Sathasivam K, Bobbs C, Turmaine M, et al. Formation of poly-glutamine insclusions in non-CNS tissue. Hum Mol Genet 1999;8:813–822.

11. Andreassen DA, Dedeoglu A, Stanojevic V, et al. Huntington’sdisease of the endocrine pancreas: insulin deficiency and diabetesmellitus due to impaired insulin gene expression. Neurobiol Dis2002;11:410–424.

12. Servadio A, Koshy B, Armstrong D, Antalffy B, Orr HT, ZoghbiHY. Expression analysis of the ataxin-1 protein in tissues fromnormal and spinocerebellar ataxia type 1 individuals. Nat Genet1995;10:94–98.

13. Chen H-K, Fernandez-Funez P, Acevedo SF, et al. Interaction ofAkt-phosphorylated ataxin-1 with 14-3-3 mediates neurodegenera-tion in spinocerebellar ataxia type 1. Cell 2003;113:457–468.

The ‘‘Imprisoned Illness:’’ MotorTic Disorder in Rainer MariaRilke’s Notebooks of Malte

Laurids Brigge

Andrea Eugenio Cavanna, MD,1,2*Maria Grazia Pattumelli,3 Tiziana Quarto,1,4

Fizzah Ali,1 and Hugh Rickards, MD1

1Department of Neuropsychiatry, University of Birminghamand BSMHFT, Birmingham, United Kingdom; 2Sobell

Department of Movement Disorders, Institute of Neurology,London, United Kingdom; 3La Sapienza University

Medical School, Rome, Italy; 4Department ofNeuroscience, University of Bari, Italy

Abstract: Rainer Maria Rilke’s novel The Notebooks ofMalte Laurids Brigge contains a reference of interest forthe catalog of literary portrayals of tiqueurs. In this arti-cle, we report his description of a Parisian character dis-playing multiple motor tic symptoms, along with a briefcommentary. � 2010 Movement Disorder Society

Key words: motor tic disorder; tics; Tourette syndrome;Rainer Maria Rilke; The Notebooks of Malte Laurids Brigge

Tic disorders of varying severity are increasingly

recognized as relatively common neuropsychiatric con-

ditions and have not escaped writers’ quest for inspira-

tion.1 To date, a number of literary portrayals of sub-

jects presenting with tic symptoms have been

reported.2,3 Moreover, James Boswell’s biography of

Dr. Samuel Johnson4 contains overt literary elements,

even though this account is based on a true person. In

all cases, the characters depicted displayed multiple

motor and vocal tics, thus suggesting a diagnosis of

Tourette syndrome. In this article, we show how Ger-

man writer Rainer Maria Rilke (1875–1926) docu-

mented the clinical features of severe motor tic disor-

der in his semiautobiographical novel, The Notebooksof Malte Laurids Brigge, published in 1910:5

*Correspondence to: Dr. Andrea Eugenio Cavanna, Department ofNeuropsychiatry, Birmingham and Solihull Mental Health NHSFoundation Trust, Barberry Building, Birmingham B15 2FG, UK.E-mail: [email protected]

Potential conflict of interest: Nothing to report.Received 14 April 2009; Revised 12 November 2009; Accepted 30

March 2010Published online 28 July 2010 in Wiley Online Library


1980 A.E. CAVANNA ET AL.


I expected that as soon as I had a better view I would see

some extraordinary and striking figure; but it turned out that

there was no one in front of me except [. . .] a tall, emaci-

ated man in a dark coat, with a soft black hat on his short,

faded-blond hair. I was sure there was nothing laughable

about this man’s clothing or behaviour, and was already try-

ing to look past him down the boulevard, when he tripped

over something. Since I was walking close behind him I was

on my guard, but I came to the place there was nothing

there, absolutely nothing. We both kept walking, he and I,

with the same distance between us. [. . .] Now there was an

interception; the man in front of me hopped down from the

sidewalk on one leg, the way children, when they are happy,

will now and then hop or skip as they walk. On the other

side of the street, he simply took one long step up onto the

side walk. But almost immediately he raised one leg slightly

and hopped on the other, once, quite high, and then again

and again. This time too you might easily have thought the

man had tripped over some small object on the corner, a

peach pit, a banana peel, anything; and the strange thing was

that he himself seemed to believe in the presence of an ob-

stacle: he turned around every time and looked at the offend-

ing spot with that half-annoyed, half-reproachful expression

people have at such moments. Once again some intuition

warned me to cross the street, but I didn’t listen to it; I con-

tinued to follow this man, concentrating all my attention on

his legs. I must admit I felt very relieved when for about

twenty steps the hopping didn’t recur; but as I looked up I

noticed that something else had begun to annoy the man.

[. . .] His coat collar had somehow popped up; and as hard

as he tried to fold it back in place, forced with one hand,

then with both at once, it refused to budge. This kind of

thing can happen. It didn’t upset me. But then I saw, with

boundless astonishment, that in his busy hands there were

two distinct movements: one a quick, secret movement, that

flipped up the collar, while the other one, elaborate, pro-

longed, exaggeratedly spilled out, was meant to fold it back

down. This observation disconcerted me so greatly that two

minutes passed before I recognised in the man’s neck,

behind his hunched-up coat and his nervously scrambling

hands, the same horrible, bisyllabic hopping that had just left

his legs. From this moment I was bound to him. I saw that

the hopping was wandering through his body, trying to break

out here or there. I understood why he was afraid of people,

and I myself began to examine the passersby, cautiously, to

see if they noticed anything. A cold twinge shot down my

spine when his legs suddenly made a small, convulsive leap;

but no one had seen it, and I decided that I would also trip

slightly if anyone began to look. That would certainly be a

way of making them think there had been some small, im-

perceptible object on the side walk, which both of us had

happened to step on. [. . .] I forgot to mention that he had a

cane [. . .] In his anxious searching he had hit upon the idea

of holding this cane against his back, at first with one hand

[. . .] right along his spine, pressing it firmly into the small

of his back and sliding the curved end under his collar [. . .]

At the next intersection two hops escaped, two small, half-

suppressed hops, but they didn’t amount to anything; and the

one really visible leap was so skilfully timed [. . .] that therewas nothing to be afraid of. Yes, everything was still all

right; from time to time his other hand too seized the cane

and pressed it in more firmly, and right away the danger was

again overcome. [. . .] I knew that as he walked and with in-

finite effort tried to appear calm and detached, the terrible

spasms were accumulating inside his body; I could feel the

anxiety he felt as the spasms grew and grew, and I saw how

he clutched his cane when the shaking began inside him.

[. . .] Several times we were caught between two carriages,

and then he would take a breath and relax a bit, and there

would be a bit of hopping and nodding. Perhaps that was the

trick by which the imprisoned illness hoped to subdue him.

[. . .] His left hand gently let go of the cane, and rose so

slowly that I could see it tremble in air. He pushed his hat

back slightly and drew his hand across his brow. He turned

his head slightly, and his gaze wobbled over sky, houses,

and water, without grasping a thing. And then he gave in.

The cane was gone, he stretched out his hands as if he were

trying to fly, and some kind of elemental force exploded

from him and bent him forward and dragged him back and

made him keep nodding and bowing and flung a horrible

dance out of him into the midst of the crowd.

DISCUSSION

Rilke’s description of a Parisian tiqueur is both

accurate and informative. We are unaware of any pre-

vious mention of this in the medical literature. The tic

symptoms, along with their body locations, are sum-

marized in Table 1. The clinical features presented by

Rilke do not fulfill current diagnostic criteria for Tour-

ette syndrome because there is no mention of any

vocal or phonic tic.6 However, the multiple motor tics

are of particular interest for their frequency, complex-

ity, and severity. Interestingly, Rilke’s description cap-

tures aspects of tic generation and tic expression which

are central to modern scientific literature. These

include feelings of mounting inner tension (‘‘premoni-

TABLE 1. Tic symptoms presented in Rilke’s description ofa Parisian tiqueur5

Simple tics Complex tics

Head Head turning GazingNodding

Arms Stretching Adjusting collarPressing cane into backSliding cane under collar

Legs Leg raising HoppingLeaping

Trunk Bending forward Bowing

1981MULTIPLE MOTOR TIC SYMPTOMS


tory urges’’ or ‘‘sensory tics’’) temporarily relieved by

tic expression7 and the social stigma elicited by dem-

onstration of tic symptoms in public spaces and cam-

ouflaging techniques adopted by individuals with

chronic tics.8

Acknowledgments: We wish to thank Tourettes Action,UK for continuing support.

Financial Disclosures: None.

Author Roles: Research project: conception (AEC, MGP),organization (AEC), execution (AEC, TQ). Manuscript: writ-ing of the first draft (AEC, TQ), review and critique (MGP,FA, HR).

REFERENCES

1. Robertson MM, Cavanna AE. Tourette syndrome: the facts.Oxford: Oxford University press; 2008.

2. Hurst MJ, Hurst DL. Tolstoy’s description of Tourette syndromein Anna Karenina. J Child Neurol 1994;9:366–367.

3. Kaplan RE. Tourette’s syndrome in George Eliot’s Silas Marner.J Neuropsy Clin Neurosci 1998;10:367.

4. Murray TJ. Dr Samuel Johnson’s movement disorder. Br Med J1979;1:1610–1614.

5. Rilke R.M. The notebook of Malte Laurids Brigge [transl. by S.Mitchell]. New York: Limited Editions Club; 1987.

6. American Psychiatric Association. Diagnostic and statistical man-ual of mental disorders, Fourth ed., Text Revision (DSM-IV-TR).Washington, DC: American Psychiatric Press; 2000.

7. Prado HS, Rosario MC, Lee J, Hounie AG, Shavitt RG, Miguel EC.Sensory phenomena in obsessive-compulsive disorder and tic disor-ders: a review of the literature. CNS Spectr 2008;13: 425–432.

8. Davis KK, Davis JS, Dowler L. In motion, out of place: the publicspace(s) of Tourette syndrome. Soc Sci Med 2004;59:103–112.

Prevalence of THAP1 SequenceVariants in German Patients with

Primary Dystonia

Anne S. Sohn, PhD,1 Nicola Glockle,1

Andrea Duarte Doetzer,1 Gunther Deuschl, MD,2

Ute Felbor, MD,3 Helge R. Topka, MD,4

Ludger Schols, MD,5 Olaf Riess, MD,1

Peter Bauer, MD,1 Ulrich Muller, MD,6

and Kathrin Grundmann, MD1*

1Department of Medical Genetics, Institute of HumanGenetics, University of Tuebingen, Tubingen, Germany;2Department of Neurology, University of Kiel, Kiel,Germany; 3Institute of Human Genetics, University of

Greifswald, Greifswald, Germany; 4Department of Neurologyand Clinical Neurophysiology, Academic Hospital

Bogenhausen, Technical University of Munich, Munich,Germany; 5Department of Neurodegenerative Diseases,Hertie-Institute for Clinical Brain Research, University of

Tuebingen, Germany; 6Institute of Human Genetics,Justus-Liebig-University, Giessen, Germany

Abstract: Primary dystonias are a clinically and geneticallyheterogeneous group of movement disorders, but only fortwo of them, i.e., dystonia 1 and dystonia 6, the disease caus-ing gene has been identified. Dystonia 1 is characterized byan early onset and is caused by a mutation in the TOR1Agene. Only recently, mutations in THAP1 have been shown tobe the cause of DYT6 dystonia. We analyzed 610 patientswith various forms of dystonia for sequence variants in theTHAP1 gene by means of high resolution melting to delineatethe prevalence of sequence variants and phenotypic variabili-ty. We identified seven sequence variants in patients and onesequence variant in a control. The sequence variants werenot detected in 537 healthy controls. Four patients presentwith generalized dystonia with speech involvement of earlyonset, another three patients suffered exclusively from cervi-cal dystonia of adult onset. These findings suggest thatTHAP1 sequence variations seem to be associated with differ-ent ages of onset and distribution of symptoms. Conse-quently, the phenotypic spectrummight be broader than pre-viously assumed. � 2010 Movement Disorder Society

Key words: dystonia; THAP1; high-resolution melting;DYT6

Additional Supporting Information may be found in the online ver-sion of this article.

The first two authors contributed equally to this work.*Correspondence to: Kathrin Grundmann, Institute of Human

Genetics, University of Tubingen, Calwerstrasse 7, D-72076 Tubin-gen, Germany. E-mail: [email protected]

Potential conflict of interest: Nothing to report.Received 28 January 2010; Revised 15 March 2010; Accepted 31

March 2010Published online 28 July 2010 in Wiley Online Library



1982 A.S. SOHN ET AL.

Dystonias are a heterogeneous group of movement

disorders characterized by sustained muscle contrac-

tions and abnormal postures.1 They can be subclassi-

fied in primary and secondary forms. Many primary

forms have a genetic etiology and can be subdivided

into ‘‘pure’’ forms, dystonia plus syndromes, and par-

oxysmal dystonias. To date at least 17 distinct types of

monogenic primary dystonias have been recognized.

Six of them are ‘‘pure’’ forms, where dystonia is the

sole or main symptom. The disease gene has been

identified in two of them, i.e., dystonia 1 (locus

DYT1) and dystonia 6 (locus DYT6). Both are inher-

ited as autosomal dominant traits.2 In both dystonia 1

and dystonia 6, onset is commonly during childhood or

adolescence. However adult-onset has been described

in both conditions as well.3,4 The disease gene in dys-

tonia 1, i.e., TOR1A was identified more than a decade

ago5 and one mutation within this gene (GAG deletion)

accounts for the majority of monogenic ‘‘pure’’ pri-

mary dystonias. More recently, the gene THAP1 was

found to be mutated in dystonia 6.6 Extensive mutation

analyses in various populations have demonstrated that

cranial and cervical involvement are common in

patients with THAP1 mutations.7–11 We performed

comprehensive mutation analysis of THAP1 in a large

cohort of German patients with primary dystonia in

order to determine the prevalence of dystonia 6 and to

further delineate its phenotype.

PATIENTS AND METHODS

Recruitment of Patients and Controls

610 patients with dystonia were studied. They were

recruited at neurological centers in Germany and clini-

cally diagnosed by specialists according to current cri-

teria.12 Patients were divided into two groups. Group I

(n 5 193) included patients with focal, segmental,

multifocal, or generalized dystonia and absence of

additional signs and symptoms. Age of onset, site of

onset, disease duration, and family history were estab-

lished from patients’ self-reports and clinical records.

Demographic data are presented in Table 1. Patients of

group II (n 5 417) presented with different forms of

pure dystonia and dystonia plus. Additional symptoms

suggestive of secondary dystonia were excluded by

neurological examination. In patients of both groups

mutations in the genes TOR1A (DYT1) and GCH1(DYT5) had been excluded (for details see Supporting

Information). All patients were of German origin. The

537 controls included 348 CEPH samples13 and 189

Germans without neurological disorders.

This study was approved by the local ethics commit-

tee. After obtaining informed consent, blood samples

were drawn for DNA extraction.

Genetic Analysis

PCR and HRM of the coding exons of THAP1 were

performed on LightCycler 480 (Roche Diagnostics,

Germany) using LightCycler1 480 HRM Master

according to the manufacturer’s instructions (Roche

Applied Science, Germany). Samples showing diver-

gent HRM profiles were sequenced on both strands

(for details see Supporting Information).

RESULTS

THAP1 sequence variants were found in 4 patients

of Group 1 (2%) and in three subjects of Group 2

(0.7%; Supporting Information Figure 1). The overall

frequency of THAP1 sequence variants was 1.14% (7/

610). None of the patients with a THAP1 sequence

variants had a positive family history. Clinical findings

and THAP1 sequence variants are given in Table 1.

The sequence variants comprised five missense muta-

tions, one 2-bp deletion, and one nonsense mutation.

All are located in the coding region of THAP1 (Fig. 1)

and were not discovered in any of the 537 European

controls. An additional sequence change, a one 2-bp

deletion (c.197_198delAG) was discovered in a control

subject (Supporting Information Figure).

THAP1 is a DNA-binding protein which contains an

N-terminal THAP domain, a zinc finger domain, a cen-

tral proline-rich region, a coiled-coil region and a bi-

partite nuclear localization sequence in its C-terminal

half (Fig. 1). The sequence variant p.H57N

(c.169C>A) is located in the zinc finger domain of the

THAP1 protein. It affects a highly conserved histidine

at position 57, which forms a zinc binding site together

with three cysteine residues. Apart from generalized

dystonia, the patient presented with a mild speech dis-

ability (Table 2). The sequence variant p.Y137C

(c.410A>G) is located 3 amino acids upstream of the

nuclear localisation signal. The patient developed oro-

bulbar symptoms during pregnancy followed by cervi-

TABLE 1. Characteristics of the study populationof Group I

Type ofdystonia

No. ofpatients

Sexmale/female

Mean 6 SDAge at onset

Family historyPositive no. (%)

Generalized 14 6/8 16 6 11 1 (7)Multifocal 8 4/4 22 6 12 1 (12)Segmental 28 11/17 42 6 18 3 (11)Focal 143 53/90 47 6 14 6 (4)

1983THAP1 SEQUENCE VARIANTS IN PRIMARY DYSTONIA


cal dystonia (Table 2). The sequence variant p.Q124X

(c.370C>T) produces a stop codon immediately before

the nuclear localization sequence. Symptoms in the

patient started in the lower limbs during early child-

hood. Cranial symptoms manifested as Meige syn-

drome, anterocollis, and pharyngolaryngeal dystonia

(Table 2). The sequence variant c.388_389delTC is

also located immediately before the nuclear localiza-

tion sequence and results in an early stop codon

(p.S130fsX133). The patient suffered exclusively from

primary cervical dystonia (Table 2). The remaining

three sequence variants [c.427A>G, (p.M143V),

c.574G>A (p.D192N), c.247T>C (p.C83R)] were

detected in patients presenting with cervical dystonia

as the only clinical sign. Age of onset was 19, 46, and

30 years of age and none of these patients developed

other dystonic symptoms during a course of several

years (Table 2). These sequence changes affect differ-

ent regions of the gene. The p.M143V (c.427A>G)

sequence variant is located in the coiled-coil region of

the protein, the p.D192N (c.574G>A) sequence variant

in the C-terminal end of the protein, and the pC83R

(c.247T>C) sequence variant affects an amino acid ad-

jacent to the THAP domain.

DISCUSSION

This study describes 7 THAP1 sequence variants in

patients with primary dystonia. All sequence variations

except c.388_389delTC have not been recognized in a

patient before.7–11 The overall frequency of sequence

variations was �1%, and corresponds to the frequency

reported in previous studies.7–9 The sequence variations

are spread over the entire gene and affect among other

regions the THAP and the coiled-coil domain (Fig. 1).

Regarding the clinical manifestation, 3 of 7 patients

(patient 1, c.169C>A; patient 2, c.410A>G; patient 3,

c.370C>T; Table 2), presented with a phenotype con-

sistent with the typical phenotype described for dysto-

nia 6 starting early in life with involvement of the cra-

niocervical region and affection of pharyngolaryngeal

or orobulbar muscles.14 Of note, the other four patients

suffered exclusively from cervical dystonia (Table 2).

The mean age of onset of 32.5 years is earlier than

reported for the classical craniocervical dystonia,

which has a mean age of onset of 54.3 years.15

The sequence changes reported are the likely cause

of dystonia in these patients. None of these changes

was found in any of the 537 controls; two sequence

changes (p.Q124X and p.S130fsX133) caused a stop

codon and thus truncation of important functional

domains of the THAP1 protein; the c.169C>A variant

(p.H57N) abolishes DNA- and zinc-binding activity in

vitro;16,17 and the c.388_389delTC variant has previ-

ously been reported in another dystonia patient with

early onset (9 years) who presented with speech prob-

lems followed by writer’s cramp.9

We identified one sequence change (c.197_198delAG)

in a German control that results in truncation of

THAP1 (p.E66fsX84). As the controls studied are

anonymous we were not able to retrace the mutation.

The mutation carrier might have shown very mild

symptoms, which would not have been reported. A

pathologic phenotype might also have been absent

due to reduced penetrance which has been reported in

dystonia 6.14

Given variable expression and reduced penetrance in

dystonia 6, additional genetic and environmental fac-

tors appear to contribute to disease manifestation and

severity of signs and symptoms. In fact, variable

expression and reduced penetrance might explain ab-

sence of a positive family history in all mutation car-

riers reported here. Unfortunately, this possibility could

not be tested experimentally since parents were not

available for clinical examination or mutation analysis.

In this study, we used HRM for mutational analy-

sis. This technique might not detect homozygous

FIG. 1. Scheme of the THAP1 protein. The THAP domain is shown in orange, the proline rich region in green. The nuclear localization sitewithin the coiled-coil domain (blue) is shown in red. All identified sequence variations are shown (arrows). [Color figure can be viewed in theonline issue, which is available at wileyonlinelibrary.com.]


1984 A.S. SOHN ET AL.

mutations, exon rearrangements, whole exon dele-

tions, and intronic changes. Nonetheless, frequency

of mutations detected is in concordance with pub-

lished data.7–9

In conclusion, the findings in the large cohort of

patients studied confirm previous observations of pref-

erential cranio-cervical involvement in dystonia 6 and

emphasize the importance of THAP1 mutation analysis

in patients with cranio-cervical involvement, affection

of speech, and tendency to generalize. Our study also

extends the phenotypic spectrum associated with

THAP1 sequence variations, as we identified THAP1sequence variations in patients with focal cervical dys-

tonia. The clinical manifestation of cervical dystonia in

DYT6 mutation carriers has also been reported by Xiao

et al. 2009 who identified THAP1 sequence variations

in a cohort of mainly adult-onset primary dystonia.11

Consequently THAP1 sequence variations seem to be

associated with different ages of onset and distribution

of symptoms and thus the phenotypic spectrum might

be broader than previously assumed.

Author Roles: A. Sohn: conception and design, executionof the project, data analysis; N. Glockle: conception anddesign, execution of the project, data analysis; A. Duarte Dot-zer: execution of the project; L. Schols: data acquisition; G.Deuschl: data acquisition; U. Felbor: data acquisition; H.Topka: data acquisition; U. Muller: data acquisition and editingand revising of the text; P. Bauer: conception and design, dataanalysis, revising of the text; O. Riess: conception and design,data analysis, revising of the text; and K. Grundmann: concep-tion and design, data analysis, drafting, editing and revising ofthe text.

Financial Disclosures: Anne S. Soehn: Grants—Fortuenefoundation; Employment—Government employee. N.Gloeckle: Employment—Government employee. AndreaDuarte Doetzer: Grants—received funding from CNPq(n:155 61 2108 9000); Employment—Government em-ployee. G. Deuschl: Honoraria—Medtronic, Lundbeck, Teva;Grants—German Research Council, German Ministry of Edu-cation and Research, Medtronic; Employment—Governmentemployee; Royalties—Thieme publisher. U Felbor: Grants—UF is supported by the Bavarian Genome Network (Bay-Gene) and the Deutsche Forschungsgemeinschaft/Graduier-tenkolleg 1048; Employment—Government employee. L.Schols: Grants—has received funding from the EU forEUROSCA (LSHM-CT-2004-503304), the Bundesministie-rium fur Bildung und Forschung for RISCA (01GM0820),EUROSPA (01GM0807), LEUKONET (01GM0838) andmitoNET (01GM0864) as well as from the Deutsche For-schungsgemeinschaft (SCHO754/4-1) in the last 12 months;Employment—Government employee. O. Riess: AdvisoryBoards—Centogene; Grants—funding from the EU forEUROSCA (LSHM-CT-2004-503304), RATstream (037846),GENEPARK (037544), Mitotarget (HEALTH-F2-2008-223388), Neuromodel (215618-2), NEURASYN (238316),Techgene (223143), MarkMD (230596) the Bundesministie-rium fur Bildung und Forschung for RISCA (01GM0820),EUROSPA (01GM0807), NGFNplus ‘‘Parkinson’’ (01GS08134)and MRNET (01GS08162) as well as from the Deutsche For-schungsgemeinschaft (RI 682/10-1); Employment—Govern-ment employee. P. Bauer: Advisory Boards—Centogene;Honoraria—Roche Diagnostics; Grants—research grants ofthe German Research Council (BMBF) to GeNeMove(01GM0603), EUROSPA (01GM0807) and RISCA(09GM0820) as well as from the EU for EUROSCA (LSHM-CT-2004-503304), MarkMD (FP7-People PIAP-2008-230596) and TECHGENE (FP7-Health 2007-B 223143);received funding from the HSP-Selbsthilfegruppe Deutsch-land; Employment—Government employee. U. Muller:Grants—received funding from the PSP-Society; Employ-

TABLE 2. List of THAP1 sequence variations identified by HRM in a cohort of 610 dystonia patients

Case Study group Sex Clinical diagnosis Exon Nucleotide change Effect on protein level

1 II f Generalized dystonia 2 c.169 C>A p.H57NStarting in lower limbMild dysarthriaOnset in early childhood

2 II f Cervical dystonia, Meige syndrome(oro-mandibular dystonia),pharyngolaryngeal dystonia

3 c.410A>G p.Y137C

3 II Generalized dystonia 3 c.370C>T p.Q124XStarting in lower limbMeige syndrome (oro-mandibular dystonia),Onset during pregnancy

4 I f Cervical dystonia 3 c.388_389delTC p.S130fsX133Onset at age 35

5 I f Cervical dystonia 3 c.427A>G p.M143VOnset at age 46

6 I m Cervical dystonia 3 c.574G>A p.D192NOnset at age 19

7 I f Cervical dystonia 2 c.247T>C p.C83ROnset at age 34

8 II f Control, no symptoms of dystonia reported 2 c.197_198delAG p.E66fsX84

1985THAP1 SEQUENCE VARIANTS IN PRIMARY DYSTONIA


ment—Government employee. K. Grundmann: Grants—hasreceived funding from the Dystonia Medical Research Foun-dation; Employment—Government employee.

REFERENCES

1. Fahn S. Concept and classification of dystonia. Adv Neurol 1988;50:1–8.

2. Muller U. The monogenic primary dystonias. Brain 2009;132(Part 8):2005–2025.

3. Almasy L, Bressman S, de L, Risch N. Ethnic variation in theclinical expression of idiopathic torsion dystonia. Mov Disord1997;12:715–721.

4. Grundmann K, Laubis-Herrmann U, Bauer I, et al. Frequencyand phenotypic variability of the GAG deletion of the DYT1gene in an unselected group of patients with dystonia. Arch Neu-rol 2003;60:1266–1270.

5. Ozelius LJ, Hewett JW, Page CE, et al. The early-onset torsiondystonia gene (DYT1) encodes an ATP-binding protein. NatGenet 1997;17:40–48.

6. Fuchs T, Gavarini S, Saunders-Pullman R, et al. Mutations in theTHAP1 gene are responsible for DYT6 primary torsion dystonia.Nat Genet 2009;41:286–288.

7. Bonetti M, Barzaghi C, Brancati F, et al. Mutation screening of theDYT6/THAP1 gene in Italy. Mov Disord 2009;24:2424–2427.

8. Bressman SB, Raymond D, Fuchs T, et al. Mutations in THAP1(DYT6) in early-onset dystonia: a genetic screening study. LancetNeurol 2009;8:441–446.

9. Djarmati A, Schneider SA, Lohmann K, et al. Mutations in THAP1(DYT6) and generalised dystonia with prominent spasmodic dyspho-nia: a genetic screening study. Lancet Neurol 2009; 8:447–452.

10. Houlden H, Schneider SA, Paudel R, et al. THAP1 mutations(DYT6) are an additional cause of early-onset dystonia. Neurology2010;74:846–850.

11. Xiao J, Zhao Y, Bastian RW, et al. Novel THAP1 sequence var-iants in primary dystonia. Neurology 2010;74:229–238.

12. Fahn S, Eldridge R. Definition of dystonia and classification of thedystonic states. Adv Neurol 1976;14:1–5.

13. Dausset J, Cann H, Cohen D, et al. Centre d’etude du polymor-phisme humain (CEPH): collaborative genetic mapping of thehuman genome. Genomics 1990;6:575–577.

14. Saunders-Pullman R, Raymond D, Senthil G, et al. Narrowing theDYT6 dystonia region and evidence for locus heterogeneity in theAmish-Mennonites. Am J Med Genet A 2007;143A:2098–2105.

15. Defazio G, Abbruzzese G, Girlanda P, et al. Does sex influenceage at onset in cranial-cervical and upper limb dystonia? J NeurolNeurosurg Psychiatry 2003;74:265–267.

16. Bessiere D, Lacroix C, Campagne S, et al. Structure-function anal-ysis of the THAP zinc finger of THAP1, a large C2CH DNA-bind-ing module linked to Rb/E2F pathways. J Biol Chem 2008;283:4352–4363.

17. Clouaire T, Roussigne M, Ecochard V, Mathe C, Amalric F, Gi-rard JP. The THAP domain of THAP1 is a large C2CH modulewith zinc-dependent sequence-specific DNA-binding activity. ProcNatl Acad Sci USA 2005;102:6907–6912.

Specific Pattern of EarlyWhite-Matter Changes in PureHereditary Spastic Paraplegia

Thomas Duning, MD,1*Tobias Warnecke, MD,1

Anja Schirmacher, PhD,1Hagen Schiffbauer, MD,2

Hubertus Lohmann, PhD,1Siawoosh Mohammadi, PhD,1

Peter Young, MD,1 and Michael Deppe, PhD1

1Department of Neurology, University Hospital of Munster,Munster, Germany; 2Department of Clinical Radiology,

University Hospital of Munster, Munster, Germany

Abstract: Hereditary spastic paraplegias (HSP) are geneti-cally and clinically heterogeneous neurodegenerative dis-orders. Most MR studies on HSP include very heterogene-ous samples of patients, and findings were inconsistent.Here, we examined six patients with pure HSP and SPG4mutations by clinical evaluation, detailed neuropsychologi-cal testing, and neuroimaging analyses, including conven-tional MRI, diffusion tensor imaging (DTI), and brainvolumetry. Differences of voxel-wise statistics and ROI-based analysis of DTI data between patients and 32healthy volunteers were evaluated. Although conventionalMRI and brain volumetry were normal, DTI revealedwidespread disturbance of white matter (WM) integrity(P < 0.001), mainly affecting the corticospinal tract. Withlonger disease duration, frontal regions were alsoinvolved. The WM changes were also present in subclini-cal subjects harbouring the pathogenic mutation. Thesesubtle WM abnormalities have functional relevancebecause they correlated with clinical symptoms. Thus,early alterations of nerve fibres, which can be detected byDTI, might serve as a biological marker in HSP, in par-ticular with respect to future longitudinal studies. � 2010Movement Disorder Society

Key words: hereditary spastic paraplegia; diffusion tensorimaging; white-matter changes; SPG4

Hereditary spastic paraplegia (HSP) is a clinically

and genetically heterogeneous neurodegenerative disor-

der characterized by progressive spasticity and weak-

ness of the lower limbs occurring in the absence (pure

HSP) or presence (complicated HSP) of additional

The first two authors contributed equally to this work.*Correspondence to: Dr. Thomas Duning, MD, Department of

Neurology, University Hospital of Muenster, Albert-Schweitzer-Str.33, 48149 Muenster, Germany. E-mail: [email protected]

Potential conflict of interest: Nothing to report.Received 21 December 2009; Revised 11 March 2010; Accepted



1986 DUNING ET AL.


major clinical features such as cognitive impairment

and extrapyramidal or cerebellar symptoms.1 To date,

45 genetic loci (SPG1-45) have been mapped and 17

genes identified.2,3

In the diagnostic work-up of HSP, magnetic reso-

nance imaging (MRI) of the brain has been mainly

used to exclude other diseases, included in the differ-

ential diagnosis of HSP. Conventional MRI examina-

tions of HSP patients suggest a loss of volume in the

corpus callosum and a higher incidence of white matter

lesions in some cases.4–7 However, most MRI studies

on HSP include very heterogeneous samples of patients

with clinical or less often genetically proven HSP

forms, and results were inconsistent.1,6–8 Only a few

studies have employed advanced neuroimaging techni-

ques in the evaluation of HSP patients, e.g., diffusion

tensor imaging (DTI).9–12 DTI is a new MRI technique

sensitive to white matter microstructure integrity.13

This study combined high-resolution DTI with statis-

tical parametric mapping (SPM) to determine subtle

microstructural abnormalities across the entire brain in

patients with genetically proven pure HSP (SPG4) and

normal conventional MRI. The purpose was to identify

specific patterns of an early cerebral affection in HSP

and its association with clinical symptoms.

METHODS

Subjects

Six SPG4-HSP patients from two families (median

age 53.3 years, SD 6 14.8 years, range, 34–74 years)

were recruited from our outpatient clinic. Every subject

was examined by experienced neurologists, and a

detailed medical history was obtained. All patients per-

formed a comprehensive neuropsychological test bat-

tery (Table 1).12

The control group was composed of 32 healthy vol-

unteers (17 women, median age 52.1 years, SD 6 11.8

years, range, 36–61 years). All control subjects had a

normal neurologic examination, no evidence of neuro-

logic disease, and normal MRI. All individuals

gave written informed consent prior to examinations.

The study protocol was approved by the local ethics

committee.

MRI Acquisition and Image Analysis

Image data were obtained on a 3.0-T system (Gyro-

scan Intera, Philips Medical System) with a high-reso-

lution structural T1-weighted 3D turbo-field-echo

sequence for brain volumetry (field-of-view of 25.6 320.5 3 16 cm3, reconstructed after zero filling to 512

3 410 3 320 cubic voxels with an edge length of 0.5

mm), and T2-weighted and FLAIR imaging. For DTI,

we employed echo planar imaging (EPI) with 20 diffu-

sion directions (two b-factors, 0 s/mm2 and 1000 s/

mm2, TR 5 9.8 s/TE 5 98 ms, voxel size: 0.89 mm

3 0.89 mm 3 3.6 mm, 2 averages).

The diffusion tensor and fractional anisotropy (FA)

maps (FA value of each voxel) were calculated from

spatially normalized images. The method has been

described in detail previously.14 Voxel-based analysis

of the FA image was performed using SPM5 (Well-

come Department of Imaging Neuroscience, London).

Differences of FA values between the patients and con-

trols were statistically evaluated by analysis of covari-

ance, modeling the factor age as co-variable to account

for the age dependency of the FA (P < 0.001). Addi-

tionally, the linear regression tool of the software was

employed to correlate maps of decreased FA with dis-

ease duration. After controlling for age, statistical

threshold for the correlation analysis was set at P 50.01 for multiple comparisons (FWE).

To assess the magnitude of regional FA alterations,

we also performed additional quantitative region-of-in-

terest (ROI) analyses. Mean FA values were calculated

within 11 defined ROIs, which were derived from an

averaged and symmetrized (x-axis) mask of the healthy

individuals with FA-values > 0.4 by deleting voxels

not associated with the respective structures. To put

the results more into perspective, FA values of the

SPG4 patients were also compared with FA values of

previously reported group of complicated HSP patients

with proven SPG7 mutations.12

Brain tissue volumes, normalized for subject head

size, were calculated from the high-resolution T1-

weighted images, using the well-established cross-sec-

tional version of the Structural Imaging Evaluation of

Normalized Atrophy (SIENA) software (SIENAx).15

Differences between patients and healthy controls were

evaluated by analysis of covariance (P < 0.05).

RESULTS

Subject Characteristics

Table 1 summarizes the detailed findings of genetic,

clinical, and neuropsychological evaluation. The indi-

viduals V and VI were not complaining about any neu-

rological symptoms, but neurological examination of

both revealed pathological hyperreflexia. The sequence

variation c.1070T > G (patient II and VI) was a novel

pathogenic SPG4 mutation. Neuropsychological evalu-

ation revealed slight to moderate disturbance of atten-

1987SPECIFIC DTI CHANGES IN PURE HSP


TABLE

1.Clinicalan

dneurop

sycholog

ical

finding

sof

thesixSP

G4-pa

tients

PatientNo.

III

III

IVV

VI

Mutationat

DNA

level

c.839_840delAG;

PTC110aa

c.1070T>G;

p.I357S

c.839_840delAG;

PTC110aa

c.839_840delAG;

PTC110aa

c.839_840delAG;

PTC110aa

c.1070

T>G;p.I357S

Sex

Male

Male

Fem

ale

Fem

ale

Fem

ale

Fem

ale

Onsetage(years)

40

37

51

42

34

38

Disease

duration(yr)

10

30

10

7<1

<1

Upper

limb

Spasticity

22

21

22

Weakness

22

22

22

Ataxia

22

22

22

Hyperreflexia

12

21

22

Sensory

impairm

ent

22

22

22

Lower

limb

Spasticity

11

111

11

11

22

Weakness

211

12

22

Ataxia

12

22

22

Hyperreflexia

11

111

11

11

11

Sensory

Impairm

ent

211

11

22

Babinskisign

Positive

Positivewithouttrigger

Positive

Positive

negative

negative

Walkingability

Unaided

Wheelchair

Unaided

Unaided

unaided

unaided

Bladder

dysfunction

111

21

22

Cognitivefunction

Notdone

Orientation

Norm

alNorm

alNorm

alnorm

alnorm

alApraxia

Norm

alNorm

alNorm

alnorm

alnorm

alMMSE

28

29

30

30

30

Attentionandworkingmem

ory

NAI Digital

symbolsubstitution

40/45%/norm

al38/60%/norm

al35/70%/norm

al30/87%/norm

al32/75%/norm

alCWIT

reading

29/30%/norm

al24/40%/norm

al29/30%/norm

al22/60%/norm

al22/60%/norm

alCWIT

colournam

ing

25/60%/norm

al24/70%/norm

al23/75%/norm

al22/75%/norm

al24/60%/norm

alWMS-R

Digital

span

forw

ard

8/53%/norm

al7/53%/norm

al9/69%/norm

al9/75%/norm

al8/57%/norm

alDigital

Span

Backward

8/71%/norm

al6/53%/norm

al7/52%/norm

al8/73%/norm

al7/58%/norm

alTrail-m

akingtest

[A]

26/70%/norm

al36/60%/norm

al28/70%/norm

al24/60%/norm

al26/50%/norm

alExecutivefunction

CWIT—

interference

condition

28/20%/close

below

average

32/14%/close

below

average

30/18%/close

below

average

13/78%/norm

al14/70%/norm

alRWT—

Letterfluency

[‘S’]

11/16%/close

below

average

14/45%/norm

al16/60%/norm

al19/65%/norm

al16/50%/norm

alTrail-m

akingtest

[B]

65/35%/norm

al110/20%/close

below

average

75/20%/close

below

average

45/80%/norm

al50/70%/norm

alVisuospatialskills

ROCF

Copy

33/16%/close

below

average

33/40%/norm

al35/60%/norm

al35/>

20%/norm

al35/>

20%/norm

alDelayed

recall

16/16%/close

below

average

15/16%/close

below

average

16/16%/close

below

average

24/62%/norm

al26/76%/norm

alVerbal

learningandmem

ory

ALVT

Recalltrial1

4/20%/close

below

average

7/70%/norm

al6/40%/norm

al8/75%/norm

al7/50%/norm

alRecalltrail5

10/30%/norm

al12/40%/norm

al14/65%/norm

al15/80%/norm

al14/60%/norm

alTotaltrials1to

534/15%/close

below

average

40/25%/norm

al50/55%/norm

al58/80%/norm

al50/40%/norm

alDelayed

recall

12/50%/norm

al11/45%/norm

al11/45%/norm

al14/80%/norm

al12/50%/norm

alRecognition(True-False

positives)

12/35%/norm

al12/35%/norm

al13/45%/norm

al15/>

65%/norm

al15/>

65%/norm

al

Ratingofcognitivefunction:test

scores/age-matched

percentile/evaluation.

Pat.V

andVI(rightbars):Clinically

notaffected

25

absent;1

5mild;11

5moderate;

111

5severe;

PTC:premature

term

inationcodon.

tion and executive function but no dementia in three

patients (I, II, and IV).

Brain Volume Differences

There were no significant differences in brain vol-

umes of the SPG4-HSP patients (gray matter, white

matter (WM), relative and normalized brain volumes)

compared with the group of healthy controls. This was

concordant with the conventional neuroradiological

reading showing no abnormalities on conventional MR

imaging.

Voxel-Based FA Analysis

Voxel-based FA analysis of the SPG4 patients

revealed significant FA reductions covering widespread

parts of the brain (P < 0.01), when compared with the

32 healthy controls. With a higher statistical threshold

(P < 0.001), the clusters with significant FA changes

were mainly confined to the temporal lobes and the

corticospinal tracts (Fig. 1A). In addition, voxel-wise

FA analysis of the two SPG4 patients (V and VI) with

only subtle clinical affection was performed as a sepa-

rate group. Interestingly, pattern of significant

decreased FA values had a substantial similarity to the

severely affected SPG4 patients (I–IV), relative to the

healthy controls (Fig. 1B). Regression analysis demon-

strated significant clusters of negative correlation

between FA values and disease duration in corticospi-

nal fibres and also in frontal regions of the brain

(Fig. 1C).

In ROI analysis, only FA values of the occipital

lobe and the splenium did not significantly differ

between HSP patients and the control subjects. Post-

hoc t-test (P < 0.001) revealed significant FA reduc-

tion in ROIs of the corpus callosum, the genu, the tem-

poral lobes, and the whole brain of the pure HSP

(pHSP) patients, when compared with the healthy con-

trols (Fig. 2). Consistent with the additional clinical

features, FA values of the previously reported group of

complicated HSP patients (cHSP)12 were lower in all

ROIs except in the corpus callosum. However, only

FA values of the ROI covering the brainstem signifi-

cantly differed between both HSP groups (cHSP <pHSP).

DISCUSSION

Herein, we provided evidence that genetically pro-

ven HSP patients with normal conventional MRI

showed widespread white matter abnormalities on

DTI analysis. Consistent with the clinical symptoms,

in particular, the corticospinal tracts were affected.

Furthermore, we could show that voxel-based FA

analysis holds promise for detecting very early cere-

bral abnormalities in SPG4-pHSP patients with unre-

vealing conventional MRI and normal brain volumetry

and only subtle clinical affction (hyperreflexia)

(Fig. 1B).

In accordance to Tallaksen et al.,16 we also found

subtle cognitive impairment in three subjects with pure

SPG4 HSP, mainly in patients with longer disease du-

ration. Interestingly, regression analysis revealed an

association between disease duration and FA reduc-

tions in the frontal lobes. Because lesions of frontal

subcortical circuits are known to be associated with

distinct neurobehavioral syndromes, the localization of

these WM abnormalities was in accordance with the

clinical symptoms. Thus, apart from corticospinal

fibres, an involvement of frontal WM might also

explain some of the clinical symptoms that occur with

longer duration of the disease.

On ROI analysis, only the mean FA values in the

ROIs covering the occipital lobes and the splenium

were not significantly decreased. In general, ROI anal-

ysis revealed that SPG4-pHSP patients exhibited lesser

(but still significant) FA reductions compared with the

group of recently reported SPG7-cHSP patients.12

However, only the ROI covering the brainstem showed

a significant difference of FA values between both

HSP groups (SPG7-cHSP < SPG4-pHSP), whereas the

FA values of the other ROIs—including the frontal

lobes—did not significantly differ between both forms.

Alterations in two main cellular functions have been

attributed to the underlying molecular pathomechanism

of HSP. SPG4, the gene encoding for spastin, interacts

with microtubules and promotes their disassembly.

Impairment of its function leads to axonal swelling and

consecutive axonal degeneration.2 The SPG7 gene enc-

odes an ATP-dependent proteolytic complex located at

the mitochondrial inner membrane.17 Thus, SPG7

mutations primarily lead to complex HSP forms and

show some overlapping symptoms with mitochondrial

disease.1,2,12 The different pattern of DTI changes in

both HSP forms may reflect these two differing patho-

mechanistical backgrounds.

Although FA changes primarily involved the cortico-

spinal tracts, the upper extremities of SPG4 patients

are typically not affected. It has been reported that an

impaired energy supply of the axonal transport pre-

dominantly leads to a degeneration of the longest CNS

axons.8,18 The resolution of the DTI does not allow to

distinguish between corticospinal fibres of the upper or

lower extremities. However, with regard to the clinical

1989SPECIFIC DTI CHANGES IN PURE HSP


FIG. 1. SPM ‘‘glass brain’’ representation (left) and coronal/axial/sagittal slices (right) of voxels with a significant decrease in fractional anisot-ropy (FA) among all HSP patients (A) compared with 32 normal controls (P < 0.001, uncorrected; > 50 contiguous voxels). The statistical mapwas superimposed on an averaged FA template of the control group. Colored bars represent t-values; display threshold is set at t value > 3.42. B,When calculated as a separate group, pattern of FA changes of two SPG4 individuals (V, VI) with only slight clinical affection (B) had substan-tial similarity to the FA maps of more severely affected SPG4 patients. C, Clusters of correlation between decreased FA and disease duration (P< 0.01, > 50 continuous voxels; corrected for multiple comparison by false discovery rate method). Apart from corticospinal fibres, an involve-ment of frontal WM might also explain some of the clinical symptoms that occur with longer duration of the disease. [Color figure can be viewedin the online issue, which is available at wileyonlinelibrary.com.]

1990 DUNING ET AL.


FIG. 2. Analysis of covariance revealed significant FA differences between HSP patients and healthy controls in the ROIs shown here. Consistentwith the more severe phenotype, FA values of the complicated forms were lower in all ROIs except the corpus callosum, relative to the pure HSPforms (posthoc t-test; P < 0.001). However, only the ROI covering the brainstem showed a significant difference of FA values between both HSPgroups (cHSP < pHSP).

symptoms, the FA changes most likely reflect a micro-

structural damage of the longest motor fibres of the

corticospinal tract.

In summary, both voxel-wise FA statistics and ROI-

based FA analysis provide evidence that DTI could be

a sensitive technique to uncover subtle microstructural

cerebral abnormalities in HSP with unrevealing con-

ventional MRI and normal brain volumetry. Our data

support the notion that subtle WM changes on DTI are

the first clinicoanatomical abnormalities detectable,

before distinctive regional degeneration on conven-

tional MRI becomes visible. When compared with

complicated HSP-forms, even pure HSP patients with

less severe phenotype showed widespread WM abnor-

malities on DTI analysis, mainly affecting the cortico-

spinal tracts and temporal regions. These pattern of

WM changes where also evident in SPG4 patients with

only subtle clinical affection. Future studies may inves-

tigate whether DTI can detect preclinical degeneration

and, thus, serve as a predictive test of HSP. Further-

more, regarding its functional significance, FA changes

might serve as a biological marker of the phenotype in

HSP, in particular with respect to future longitudinal

studies.

Financial Disclosures: T.D.: Grants: Genzyme. T.W.,A.S., H.S., H.L., S.M.: none. P.Y.: Honoria: Genzyme, Boeh-ringer, Ingelheim, MEDA Pharma; Grants: University ofMunster (IMF). M.D.: Grants: Deutsche Forschungs Gemein-schaft (DFG).

Author Roles: T.D. was involved in the conception, orga-nization, and execution of the research project and in designand execution of the statistical analysis and also in writing,in the review, and critique of the manuscript. T.W. wasinvolved in the conception, organization, and execution ofthe research project and in design and execution of the statis-tical analysis and also in the writing of the first draft, review,and critique of the manuscript. A.S. was involved in the exe-cution of the research project and in execution of the statisti-cal analysis and also in the review and critique of the manu-script. S.M. was involved in the organization of the researchproject and in design of the statistical analysis and also inthe review and critique of the manuscript. H.S. and H.L.were involved in the execution of the research project andstatistical analysis and in the review and critique of themanuscript. M.D. and P.Y. were involved in the conceptionand organization of the research project and in design, execu-

tion, review, and critique of the statistical analysis and alsoin the review and critique of the manuscript.

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