ORIGINAL COMMUNICATION

Transcranial sonography in pantothenate kinase-associatedneurodegeneration

Vladimir S. Kostic • Marina Svetel •

Milija Mijajlovic • Aleksandra Pavlovic •

Milica Jecmenica-Lukic • Dusan Kozic

Received: 11 August 2011 / Revised: 12 October 2011 / Accepted: 15 October 2011 / Published online: 5 November 2011

� Springer-Verlag 2011

Abstract After it was reported that increased tissue iron

concentrations were associated with increased echogenicity

of the substantia nigra (SN) obtained with transcranial

sonography (TCS) in animal and postmortem studies, our

goal was to use this method in a disorder characterized with

iron accumulation in human brain tissue. Therefore, mag-

netic resonance imaging (MRI) and TCS were conducted in

5 unrelated patients with pantothenate kinase-associated

neurodegeneration (PKAN), caused by PANK2 mutations.

All patients had an eye of the tiger sign. Hypointense

lesions on the T2-weighted MRI images were restricted to

the globus pallidus (GP) and SN. TCS also revealed

bilateral hyperechogenicity restricted to the LN and SN,

with normal DTV values. Both TCS and MRI studies in

PKAN patients are in accordance with the pathological

findings that accumulation of iron, even in advanced cases,

is restricted to the GP and SN, suggesting selective

involvement of these structures.

Keywords Transcranial sonography � Pantothenate

kinase-associated neurodegeneration

Introduction

From the first report of an abnormality on transcranial

sonography (TCS) that was specific for Parkinson’s disease

(PD) (i.e., hyperechogenicity of the substantia nigra (SN)

in up to 90% of patients with PD, but also in approximately

10% of the healthy subjects) [1–3], this technique has

gathered increasing attention in other degenerative disor-

ders, because it enabled studies of not only midbrain

structures, including echogenicity of the thalamus, lentic-

ular nucleus (LN), and caudate nucleus, but also the

transverse diameter of the third ventricle and of the frontal

horn of the lateral ventricle [4, 5]. The reason for hyper-

echogenicity of the SN in PD patients is not clear, although

several reports found an association between increased iron

content in the SN and its hyperechogenicity [6–10].

Neurodegeneration with brain iron accumulation

(NBIA) encompasses a group of progressive extrapyrami-

dal disorders [11], with the pantothenate kinase-associated

neurodegeneration (PKAN) as its major form (50–70% of

cases of NBIA), caused by a mutation in the PANK2 gene

[12].

Considering the proposed role of iron in SN hyperech-

ogenicity, we investigated TCS findings in five genetically

proven cases of PKAN, a disease characterized by brain

iron deposition.

Patients and methods

Five patients with PKAN from five unrelated, nonconsan-

guineous families with clinical presentation of extrapyra-

midal symptoms and signs and neuroimaging evidence of

iron deposition in the basal ganglia were included in the

study. PANK2 mutations were identified in all our patients,

V. S. Kostic (&) � M. Svetel � M. Mijajlovic � A. Pavlovic �M. Jecmenica-Lukic

School of Medicine, Institute of Neurology CCS,

University of Belgrade, Ul. Dr Subotica 6,

11000 Belgrade, Serbia

e-mail: vladimir.s.kostic@gmail.com

D. Kozic

School of Medicine, MRI Center,

University of Novi Sad, Novi Sad, Serbia

123

J Neurol (2012) 259:959–964

DOI 10.1007/s00415-011-6294-4

as previously reported (Table 1) [13]. According to the

criteria of Hayflick et al. [11], only patient 4 (Table 1) had

‘‘classic’’, while the other 4 patients had an ‘‘atypical’’

form of the PKAN. Patients were clinically examined by

two independent neurologists (MS, VSK), and MRI scans

were analyzed by the same neuroradiologist (DK). The

study was approved by the Ethical Committee of the

School of Medicine University of Belgrade. After written

informed consent was obtained from the patients, detailed

clinical data were collected and TCS and MRI were con-

ducted on the same day.

TCS was performed (MM, who was blinded to the diag-

nosis and MRI findings) through the preauricular acoustic

bone windows using a color-coded phased-array ultrasound

system equipped with a 2.5 MHz transducer (ProSound

Alpha 10, Aloca, Japan). The ultrasound parameters chosen

were penetration depth of 16 cm, dynamic range 50–55 dB,

and high persistence. Image brightness, contrast, and time-

gain compensation were adjusted to obtain the best image.

Substantia nigra echogenic size measurements were per-

formed on axial TCS scans automatically after manually

encircling the outer circumference of the SN’s echogenic

area. According to the previously published criteria from our

laboratory [14], echogenic sizes of B0.19 cm2 were classi-

fied as normal, sizes of C0.25 cm2 as markedly hyperecho-

genic, and sizes in-between as moderately hyperechogenic.

Echogenicity of the lenticular nuclei, the heads of the cau-

date nuclei, and the thalami was classified as hyperechogenic

when it was more intense that the surrounding white matter

(classification was based on the more affected side). The

width of the third ventricle (DTV) was measured on a stan-

dardized diencephalic axial scanning plane and was deter-

mined by the minimum transverse diameter on axial TCS

scan (for details considering scanning planes see [15]).

Table 1 Demographic and clinical features of patients

Patient 1 2 3 4 5

Age (years) 29 37 38 16 35

Sex M F F M F

Age of disease onset (years) 18 25 20 10 13

Duration of the disease

(years)

11 12 18 6 22

Family history – – – – –

Consanguinity – – – – –

Mutation 1583C [ T

(T528M);

homozygous

1583C [ T

(T528M);

homozygous

1583C [ T

(T528M);

homozygous

1583C [ T

(T528M)/

1418del7

1583C [ T

(T528M)/

1418del7

Initial symptom(s) Lingual dystonia Speech disturbance Foor dystonia Oromandibular

dystonia

Oromandibular

dystonia

Dystonia ??? ?? ??? ??? ???

Oromandibulofaciolingual

dystonia

??? ?? ?? ??? ???

Chorea – – ? ? ?

Parkinsonism ? – ? ? ?

Cerebellar involvement – ? – – –

Gait disturbance ??? ? ?? ??? ???

Pyramidal involvement ?? ? ? ? –

Dysarthria ??? ?? ??? ??? ???

Dysphagia ??? ? ? ??? ??

Axial involvement ??? ? ?? ??? ??

Retinitis pigmentosa – – – ? –

Optic atrophy – – – ? –

Behavioral abnormalities ? ? ? ? ?

Cognitive impairment ? – ? ?? ?

Ocular movement disorders – – – – –

Apraxia of eyelid opening ? – – – –

Acanthocytosis – – – – –

Peripheral neuropathy – – – – –

? unknown or not examined, – not present, ? mild, ?? moderate, ??? severe

960 J Neurol (2012) 259:959–964

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Results

Demographic and clinical features of our patients are pre-

sented in Table 1. The mean age was 31 years (range

16–38 years), with rather long duration of the disease

(mean: 13.8 years; range: 6–22 years). The PANK2 muta-

tion 1583C [ T (T528M) was found in 8 out of 10 alleles.

This missense mutation was identified as one of the most

frequent among PKAN patients [13]. Dystonic phenomena

were the presenting symptom in 4 patients (Table 1). At

the inclusion in the study, the most prominent symptoms

and signs included dystonia, particularly of the oro-

mandibulofaciolingual and axial distribution, with gait

disturbances, dysarthria, and dysphagia, and pyramidal

affection.

TCS revealed hyperechogenicity restricted to the LN

and SN with normal DTV values. Extensive hyperechog-

enicities of the lenticular nuclei (Table 2) were bilateral

and symmetrical, with the exception of patient 1 whose

echogenic area was larger in the right LN). Four patients

had marked, bilateral hyperechogenicity of the SN with the

exception of patient 3, where we detected unilateral,

moderate hyperechogenicity (0.20 cm2) of the right SN,

due to an insufficient acoustic bone window.

All our patients had an eye of the tiger sign (Table 2 and

Fig. 1). Hypointense lesions on the T2-weighted images

were in accord with the TCS data, being restricted to the

globus pallidus (GP) and SN. Only in patient 2 hypointense

were lesions of the dentate nuclei also observed.

Discussion

The main finding of our study is that the hyperechogenic

areas detected in the TCS study correlate with the hypo-

intense regions detected during MRI examinations. With

both techniques the observed changes are restricted to the

LN/GP and the SN, regions where, even in advanced cases,

the accumulation of iron selectively occurs in PKAN

(Fig. 1) [16].

In patients with PD, TCS detects hyperechogenicity of

the SN in up to 90% of patients [1–3]. Until recently, only

increased tissue iron concentration was associated with an

increased area of echogenicity of the SN in animal and

postmortem studies [6, 7, 10, 17, 18]. Animal experiments

revealed a dose-dependent increase in SN echogenicity

after the stereotactic injection of various concentrations of

iron into the SN [17]. Zecca et al. [18] scanned postmortem

brains from normal individuals and a found positive cor-

relation between the echogenic area of the SN and the

concentration of iron, H- and L-ferritins, the main iron

storage proteins, but a negative correlation between ech-

ogenicity size and neuromelanin content in the SN. How-

ever, increased iron concentration alone probably cannot be

an explanation for the observed SN hyperechogenicity in

PD patients, and other factors, such as iron-binding pro-

teins may also have a role [4]. Mutational analyses of the

ceruloplasmin (critically involved in iron transport across

the cell membranes) gene variations in PD showed that at

least two of them (D554E and R793H) may be associated

Table 2 Findings of transcranial brain sonography (TCS) and magnetic resonance imaging (MRI) studies

Patient 1 2 3 4 5

TCS

SN-r (echogenic size in cm2) 0.39 0.42 0.2 0.43 0.54

SN-l (echogenic size in cm2) 0.39 0.45 0.17 0.66 0.51

SN asymmetry index 1.13 1.07 1.17 1.53 1.06

NL-r (echogenic size in cm2) 2.11 0.8 0.83 0.48 0.9

NL-l (echogenic size in cm2) 0.86 0.8 0.87 0.56 0.92

NCa-r (echogenic size in cm2) – – – – –

NCa-l (echogenic size in cm2) – – – – –

Thalamic hyperechogenicity – – – – –

Hyperechogenicity of red nucleus – – – – –

Raphe hypoechogenicity ? – – ? ?

DTV (mm) 5 6 4 5 4

MRI

,,Tiger eye‘‘sign ??? ?? ??? ? ??

Globus pallidus hypointensity ??? ??? ??? ?? ??

SN hypointensity ?? ? ? ? ?

Nucleus dentatus hypointensity – ? – – –

r right, l left, SN substantia nigra, NL lenticular nucleus, Nca caudate nucleus, DTV diameter of the third ventricle, – absent, ? mild,

?? moderate, ??? marked, TCS transcranial brain sonography, MRI magnetic resonance imaging

J Neurol (2012) 259:959–964 961

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with hyperechogenicity of the SN [8]. In addition to iron

content, Berg et al. [10] recently observed correlation of

SN echogenicity with the activation of microglia, known to

contain high amounts of ferritin.

We assumed that the proposed role of iron deposits in

the hyperechogenicity of distinct brain structures obtained

by TCS can be substantiated by the use of this method in

patients with disorders characterized with iron accumula-

tion in specific areas of the brain. Therefore, we studied

5 patients with genetically confirmed diagnosis of PKAN

(Table 1). Interestingly, a global increase in brain iron is

not seen in PKAN: instead, the accumulation of iron in

pathological studies is, even in advanced cases, rather

restricted to the GP and SN pars reticulata (SNr), sug-

gesting selective involvement of these structures [19].

Routine iron staining detects the metal mainly in the

microglia and macrophages, but also in scattered neurons

and extracellularly, around blood vessels [16]. In regions of

extensive iron deposition, axonal spheroid bodies, many

positive for iron, probably represent a consequence of the

defects in axonal transport or membrane integrity [20].

Pantothenate kinase is a regulator in the synthesis of free

fatty acids; therefore, it is not clear how defects in lipid

metabolism may cause iron deposition [20]. Schneider et al.

[21] suggested that even if iron deposition was not the initial

and causative factor, but rather an epiphenomenon of cell

degeneration, it probably had a perpetuating role in the

cascade of events following disease initiation. Indeed, it has

been shown that excess deposition of iron may cause neu-

ronal degeneration, gliosis, and spheroid formation [22].

MRI studies in PKAN are in accord with pathological

findings. The primary neuroimaging changes in PKAN due

Fig. 1 a T2-weighted MR image of the midbrain of patient with the

pantothenate kinase-associated neurodegeneration (PKAN), with

b sonographic images of corresponding midbrain axial sections,

showing hyperechogenicity of the SN. c Bilateral T2-weighted

images of the ‘‘eye of the tiger’’ sign, with d sonographic image of

corresponding region (arrow shows hyperechogenicity of the lentic-

ular nucleus)

962 J Neurol (2012) 259:959–964

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to high iron in basal ganglia are hypointense lesions in the

GP and SNr on T2-weighted images [23–26]. In pre-

symptomatic patients the hyperintense lesions predomi-

nate, but with disease progression, the hypointensities

appear and eventually prevail [27]. In PKAN, the deposi-

tion image pattern of bilateral symmetrical hyperintense

signals surrounded by hypointensity on T2-weighted ima-

ges (i.e., eye of the tiger sign) is highly specific and almost

pathognomonic feature (Fig. 1) [11, 25]. The central

hyperintensity is probably due to axonal swelling with

spheroid formation, gliosis, and neuronal loss and degen-

eration, while the surrounding hypointensity represents

iron deposition [24].

As expected, all our patients have an eye of the tiger

sign, with T2-weighted hypointense lesions restricted to the

GP and SN (Table 2). Only one patient also had hypoin-

tensity of the dentate nuclei. McNeill et al. [25] used T2*

and T2 fast spin echo brain MRI and found that in most

PKAN cases abnormalities were also restricted to GP and

SN and that 100% had an eye of the tiger sign (subtle in

some mildly affected patients), while in a minority of cases

hypointensity was observed in the dentate nuclei.

In continuation, our TCS data (Table 2) revealed bilat-

eral extensive hyperechogenic areas in the region of the

lenticular nuclei and SN (Fig. 1). Unfortunately, it is not

possible to visualize the pars reticulata separately by TCS:

only the whole SN can be seen. According to criteria used

in this study, in one patient with mild symmetrical MRI

hypointensity of the SN (patient 3 in Table 2), TCS

detected only unilateral SN hyperechogenicitiy. Hayflick

et al. [28] described the late appearance of radiographic

evidence for iron deposition in the SNr in PKAN. But even

then, since MRI in our patient showed bilateral changes,

this MRI/TCS mismatch may be due to a lower sensitivity

of TCS in revealing iron deposits.

Two previous studies addressed the same issue [29, 30].

The autosomal-recessive Kufor-Rakeb syndrome, induced

by mutations in the ATP13A2 gene (PARK9 locus), is

associated with the presence of iron accumulation in the

basal ganglia, placing the syndrome among disorders of

NBIAA [29]. Bruggemann et al. [29] showed that, despite

of an association of single ATP13A2 heterozygous muta-

tions with parkinsonism, the SN had a normal appearance

on TCS in all mutation carriers. The authors explained the

lack of SN hyperechogenicity in such patients by the

‘‘putative presence of different iron compounds and bind-

ing partners’’. In another study, Liman et al. [30] performed

TCS in 6 patients with a diagnosis of NBIA with

(3 patients) and without (3 patients) PKAN mutation, and

in 1 patient who was not genetically tested, but had typical

eye of the tiger sign. All of them had significantly

increased size of SN hyperechogenicity, together with the

hyperechogenicity of the nucleus rubber in most of them.

However, contrary to our data, they did not observe any

changes in the GP when compared to controls, possibly due

to the heavy dystonic movements that made it difficult to

properly obtain signal abnormalities.

Our study of patients with PKAN showed that echoge-

nicity changes of brain tissue identified by the TCS cor-

relate with the MRI findings and closely follow

pathological changes characterized by iron accumulation.

Acknowledgments This study was supported by a grant from the

Ministry of Science and Technology, Republic of Serbia (project no.

175090). VSK had full access to all of the data in the study and takes

responsibility for the integrity of the data and the accuracy of the data

analysis.

Conflict of interest The authors declare that they have no conflicts

of interest.

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