e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y 1 6 ( 2 0 1 2 ) 1 7 5e1 7 8

Official Journal of the European Paediatric Neurology Society

Original article

Transcranial ultrasound in neurodegeneration with brain ironaccumulation (NBIA)

Jan Liman a,*, Andreas Wellmer b, Kevin Rostasy c, Mathias Bahr a, Pawel Kermer a

aDepartment of Neurology, University of Gottingen, Robert-Koch-Str. 40, 37075 Gottingen, GermanybDepartment of Neurology, Hospital Ludmillenstift, Ludmillenstraße 4-6, 49716 Meppen, GermanycDepartment of Paediatrics, Division of Paediatric Neurology, Medical University of Innsbruck, Anichstr. 35, 6020 Innsbruck, Austria

a r t i c l e i n f o

Article history:

Received 21 September 2010

Received in revised form

26 May 2011

Accepted 18 July 2011

Keywords:

NBIA

TCS

Sonography

Hallervorden-Spatz-Syndrome

* Corresponding author. Tel.: þ49 551 391413E-mail address: jliman@gwdg.de (J. Lima

1090-3798/$ e see front matter ª 2011 Europdoi:10.1016/j.ejpn.2011.07.009

a b s t r a c t

NBIA/HSS is a neurodegenerative disorder associated with iron accumulation in specific

brain regions. To date, the diagnosis is obtained by typical MRI changes followed by genetic

mutation analysis. This procedure is laborious and limited to a few specially equipped

medical centres. Since transcranial sonography (TCS) is widely used for the early diagnosis

of PD in adults displaying parenchymal metal deposits, it is likely to be a reliable diagnostic

tool for the early diagnosis of NBIA.

In 7 patients with proven NBIA and 13 age-matched controls without record of neuro-

logical disease TCS was performed by an experienced ultrasound examiner. Data were

analysed by two blinded investigators regarding hyperechogenicity and size of the sub-

stantia nigra (SN). SN size and hyperechogenicity was significantly increased in patients

with NBIA compared to controls (students t-test: p < 0.001).

TCS appears to be a non-invasive and inexpensive screening technique in patients with

suspected NBIA. Performed by an experienced physician, it could enable an earlier diag-

nosis and pre-selection of patients for the MRI scan and genetic testing, which are still the

diagnostic gold standard.

ª 2011 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights

reserved.

1. Introduction NBIA was designated pantothenate kinase-associated neuro-

Neurodegeneration with brain iron accumulation (NBIA),

formerly known as Hallervorden-Spatz-Syndrome is an

orphan disease with an estimated prevalence of 1e3 per

million. Neuropathological hallmarks are abnormal iron

accumulations especially in the globus pallidus and the sub-

stantia nigra pars reticulata.1 Recently, mutations in the

pantothenate kinase 2 (PANK2) gene were identified as caus-

ative for up to 70% of all NBIA cases. Hence, this subtype of

9; fax: þ49 551 3914302.n).ean Paediatric Neurology

degeneration (PKAN).2,3

Symptoms usually develop in childhood, but later mani-

festations occur and are associated with certain mutations in

PANK2 gene.3 Affected patients initially present with gait

abnormalities or an increased “clumsiness” before developing

the typical clinical signs consisting of dysarthria, dystonia and

visual problems.4

In the “pre-MRI”-era the diagnosis of NBIA could only be

suspected by clinical features and verified post mortem by

Society. Published by Elsevier Ltd. All rights reserved.

Table 1 e List of genetic background, age and sex of thepatients examined.

age geneticbackground

sex Right SNarea[cm2]

Left SNarea[cm2]

NBIA 1 8 years PKAN M 0.18 0.11

NBIA 2 5 years non-PKAN F 0.4 0.17

NBIA 3 14 years PKAN F 0.39 0.27

NBIA 4 26 years non-PKAN M 0.28 0.34

NBIA 5 10 years PKAN M 0.32 0.22

NBIA 6 21 years non-PKAN F 0.28 0.39

NBIA 7 34 years unknown F 0.3 0.34

e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y 1 6 ( 2 0 1 2 ) 1 7 5e1 7 8176

a neuropathologist. To date, MR imaging is the gold standard

for diagnosing the disease by showing the pathognomonic

hypointensity within the globus pallidus along with high

signal intensity in the center of the globus pallidus internus

also known as “eye-of-the-tiger-sign” on T2-weighted

images.5 The diagnosis is then usually verified by PANK2

mutation screening in these patients. However, up to 30% of

cases who present with the clinical features do not have

a typical MRI and/or expected genetic findings.

In individuals with suspected NBIA MRI remains the diag-

nostic tool of choice, butpatientshave tobeanesthetized for this

procedure because of potential movement artefacts due to the

severe movement disorder and young age. Therefore MRI units

with an experienced anaesthesiologist, which is mostly

restrictedto largemedicalcentresinmajorcities,aremandatory.

According to our experience, these difficulties often prolong the

time from disease onset to definite diagnosis for years.

Since its first description by Becker et al. (1995), trans-

cranial sonography (TCS) has become a widely accepted

diagnostic tool in early and differential diagnosis of Parkin-

son’s disease (PD).6 More than 90% of PD patients exhibit an

increased area of hyperechogenicity of the substantia nigra

(SN), which in combination with clinical features is highly

characteristic for this disease.6,7 As there is some evidence

that the structural correlate of this hyperechogenicity in PD is

related to increased SN iron levels and since iron accumula-

tion in the SN also is one of the key features in NBIA, we

intended to test TCS as diagnostic tool for early and differ-

ential diagnosis of NBIA.8 In our opinion, TCS could become

a screening method for NBIA, as it is easy to use, even in non

anesthetised patients, widely available, cheap and is not

associated with any side effects.

In this study, we performed TCS in 6 patients with genet-

ically proven NBIA, one patient without genetic analysis, but

typical MRI findings and 13 age-matched controls without any

record of neurological disease or brain damage.

2. Materials and methods

2.1. Participants

Patients were recruited during the annual meeting of the

German NBIA Association “Hoffnungsbaum” in 2008. Six

patients had a diagnosis of NBIA with (3 patients) or without

PKANmutation (3 patients) prior to the study, one patient had

the diagnosis NBIA without any genetically testing, but with

typical MRI changes (eye- of ethe-tiger-sign). The age ranged

from 5 to 34 years (for overview see Table 1).

13 agematched individuals recruited from the Department

of Paediatric Cardiology, (age range 7e28 years), without any

neurological symptoms, served as controls.

This study was approved by the local ethical committee

(No. 1/1/08), all participants or their legal guardians gave

written informed consent to this study.

2.2. Procedure

Transcranial ultrasound was performed by an experienced

sonographer through the temporal bone window with

a 2.5Mhz phased-array transducer (Siemens, Acuson Sequoia

Erlangen Germany), as described previously.9 The ultrasound

parameters chosen were: penetration depth 14e16 cm,

dynamic range 50 dB, high persistence, gray scale and focus

were adjusted individually. The obtained data were recorded,

saved and analysed further without marking of any sono-

graphic structures.

In the mesencephalic plane the brainstem was visualized

as a hypoechogenic “butterfly shaped” structure (Fig. 1A).

Afterwards the data obtained were analysed a second time by

two independent examiners. Both examiners were blinded to

the subgroup (patient or control), and to the results of each

other. Planimetric measurements were performed automati-

cally, after manually encircling the outer circumference of

SN’s echogenic area. Measurement was only performed ipsi-

lateral to the ultrasound probe. Statistical analysis was per-

formed using two tailed students t-test.

3. Results

Size measurements of SN hyperechogenicity in the NBIA

group yielded a mean area of hyperechogenicity of 0,39 cm2

þ/� 0,04 cm2 on the right and 0,35 cm2 þ/� 0,04 cm2.on the

left side. Compared to age-matched controls with a mean

size of 0,12 cm2 þ/� 0,01 cm2 for the right and 0,1 cm2 þ/�0.006 cm2 for the left side, there was a highly significant

difference (t-test p < 0.001; Fig. 1B). Most NBIA patients also

showed hyperechogenicity of the Nucleus ruber (data not

shown), however we were not able to detect this feature

consistently which may be due to technical difficulties,

because of heavy dystonic movement in some patients. A

correlation between age or sex and size of the hyper-

echogenic SN could be documented neither for the NBIA nor

the control group, as the absolute number of subjects in this

study is too low to provide solid statistical data in this regard.

In our study all examined patients displayed a hyper-

echogenicity of the SN. But total numbers of examined

patients were too low to calculate a solid Positive Predictive

Value (PPV).

Analysis of the globus pallidus via TCS in patients who had

a typical “Eye-of-the-tiger-sign” inMRI scans did not show any

valid abnormalities compared to controls. As there is evidence

that the lenticular nucleus is also altered on TCS in diseases

with heavy metal accumulation in the brain, we also paid

special attention to this particular brain region.10 However, we

were not able to demonstrate any valid changes in this region.

Fig. 1 e A: Sonographic measurement of SN in NBIA patients and controls Butterfly-shaped hypoechogenic mesencephalic

brainstem. The hyperechogenic area at the anatomical site of the SN in two NBIA patients of different ages (upper row),

compared to age-matched controls (lower row). B: Planimetric measurement values in NBIA patients compared to controls

Comparison between both groups illustrated for each side separately show a highly significant difference between NBIA

patients and controls (t-test<0.001).

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4. Discussion

Our findings demonstrate a highly significant increase of SN

hyperechogenicity in patients suffering from NBIA compared

to age-matched controls (Fig. 1B). Taking into account that

abnormal iron accumulation in the SN is a key feature in NBIA

andmore severe than in PD, our results suggest that TCS could

serve as an easy to use and valid tool for the early and

differential diagnosis in patients with movement abnormali-

ties with regards to the diagnosis of NBIA. New neuropatho-

logical studies showed differences in iron accumulation with

regard to the genetic subtype of NBIA.11 Especially in PKAN

there was no hard evidence for iron deposits in the SN.

However, recent MRI studies identifiedmetal accumulation as

causative for signal changes in the SN of PKAN patients.12 This

highlights the controversy of neuropthological findings on one

hand and technical information gained e.g. by MRI on the

other hand. In our cohort, all patients regardless of their

genetic background displayed a hyperechogenictiy in the SN.

Though it is intriguing to speculate that iron accumulation

leads to the demonstrated changes in midbrain TCS, even in

Parkinson’s disease, where TCS is widely accepted as

a method for early and differential diagnosis, the patholgical

correlate of the hyperechogenicity is still matter of debate.13

Unfortunately, TCS is not able to provide diagnostic discrim-

ination between the genetic subtypes of NBIA in our study due

to the limited number of patients. To examine this issue,

a large, internationalstudy would be needed to achieve a crit-

ical number of individuals presenting with different

genotypes.

In contrast to previous reports14 where it was shown that

SN echogenicity is slightly increased within the first 6 years of

age we did not observe hyperechogenicity of the SN our

control group. However, onset of NBIA is usually found

beyond the fifth year of age andmean age in our control group

was 13,9 years, a time-point where SN hyperechogenicity is

almost decreased to adult ratios.14 Moreover, the increase of

SN hyperechogenicity in NBIA patients is much more

pronounced compared to the reported mildly increased SN

ratios of healthy patients below the age of 10 and therefore

represents a robust finding. This fact also explains our diffi-

culties in discriminating the red nucleus in the patients group

consistently. Due to the markedly enlarged area of the sub-

stantia nigra it was easy to detect even in patients with dys-

tonic movements. Another limitation of TCS in general is the

absence of a proper bone window in 10e20% of adult patients.

In our experience this limitation appears especially with

increasing age. In case of NBIA, probably due to the young age

of patients, we did not observe any restrictions regarding this

shortcoming of TCS. Surprisingly, we did not find consistent

abnormalities in the basal ganglia, namely the globus pallidus,

which in NBIA with PKAN is known to display the pathogno-

monic “Eye-of-the-tiger-sign”. This may partly be due to the

heavy dystonic movements of the patients. In those cases

where we were able to detect signal abnormalities the degree

was substantially less intense compared to the reported cases

by Walter et al. in Wilson’s disease.15 However, as the total

number of patients examined in this study is quite low, this,

as well as the possible differences between subtypes of NBIA

should be addressed in a larger study. Still, we conclude that

TCS in NBIA could serve as a screening method ahead of the

gold standard examinations. One main shortcoming of

sonography in general is the mandatory experience of the

examiner. But as midbrain sonography also has become

widely used and accepted in the early and differential diag-

nosis of Parkinson’s disease since its first description in 1995,

we do not rate this as a permanent drawback.6

The clinical importance of our observations is underlined

by the fact, that the MRI as the gold standard does not always

e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y 1 6 ( 2 0 1 2 ) 1 7 5e1 7 8178

show the “eye- of- the-tiger-sign”, as it may only appear

during disease progression or may even disappear during

course of the disease.3,16 Especially in light of at least one

report about an absent “eye-of-the-tiger-sign” in a patient

with a genetically proven PANK2 mutation,16 we think that

TCS is an interesting diagnostic tool, which could be used as

a screening method before MR-imaging and gene mutation

screens.

NBIA is not the only paediatric disease in which metal

deposition in the brain occurs e.g, copper accumulation in the

SN of subjects withWilson’s disease or metal accumulation in

early beginning spinocerebellar ataxias such as SCA 7 or 13.17

Taken together, our data suggest that TCS may be an

inexpensive and safe tool for the diagnosis of NBIA disorders

which could be used ahead of MR imaging and gene mutation

analysis in patients with an unexplained progressive move-

ment disorder.

Acknowledgements

We thank the German NBIA association “Hoffungsbaum e.V.”,

the patients and their families as well as the Department of

Paediatric Cardiology in Goettingen (Dr. Schill) for their

support. JL received assistance with travel expenses from

the German NBIA association. AW, KR, MB and PK have no

conflicts of interest.

Appendix. Supplementary data

Supplementary data related to this article can be found online

at doi:10.1016/j.ejpn.2011.07.009.

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