ORIGINAL COMMUNICATION

ANO10 c.1150_1151del is a founder mutation causing autosomalrecessive cerebellar ataxia in Roma/Gypsies

Teodora Chamova • Laura Florez • Velina Guergueltcheva •

Margarita Raycheva • Radka Kaneva • Hanns Lochmuller •

Luba Kalaydjieva • Ivailo Tournev

Received: 7 August 2011 / Revised: 29 September 2011 / Accepted: 1 October 2011 / Published online: 19 October 2011

� Springer-Verlag 2011

Abstract A recent report (Vermeer et al. in Am J Hum

Genet 87:813–819, 2010) implicated for the first time the

ANO10 gene in the genetic basis of autosomal recessive

cerebellar ataxias. One of the three described families were

Roma/Gypsies from Serbia, where the affected individuals

were homozygous for the truncating p.Leu384fs mutation

and displayed distinct phenotypic features (Vermeer et al.

in Am J Hum Genet 87:813–819, 2010). Based on the

history and population genetics of the Roma/Gypsies, we

hypothesised that p.Leu384fs could be another founder

mutation in this population, whose identification in a larger

number of genetically homogeneous patients will contrib-

ute to defining the phenotypic spectrum of the disorder.

Here, we describe additional patients from neighbouring

Bulgaria, outlining invariable ANO10-ataxia features and

confirming global intellectual decline as part of the phe-

notype resulting from complete Anactomin 10 deficit.

Keywords Autosomal recessive cerebellar ataxia �ANO10 � Phenotypic features � Gypsy founder mutations

Introduction

Autosomal recessive (AR) ataxias are a clinically and

genetically heterogeneous group of inherited progressive

neurodegenerative disorders that affect the cerebellum, the

spinocerebellar and sensory tracts of the spinal cord. These

diseases are rare, with prevalence estimated at around

5–6/100,000 [1]. They are characterized by earlier onset

[compared to their autosomal dominant (AD) counterparts],

usually before age 20 years [2], of imbalance, unsteady gait,

limb incoordination, and impairment of speech, swallowing,

and eye movements [3]. In contrast to AD forms, unravelling

the molecular background and genotype-phenotype corre-

lations of AR ataxias has proven to be more complicated,

T. Chamova, L. Florez, L. Kalaydjieva and I. Tournev have equal

contributions to the work.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00415-011-6276-6) contains supplementarymaterial, which is available to authorized users.

T. Chamova � V. Guergueltcheva � M. Raycheva � I. Tournev

Clinic of Neurology, University Hospital ‘‘Alexandrovska’’,

Sofia, Bulgaria

L. Florez � L. Kalaydjieva

Molecular Genetics Laboratory, Western Australian Institute

for Medical Research and Centre for Medical Research,

The University of Western Australia, Perth, Australia

R. Kaneva

Molecular Medicine Centre, Medical University-Sofia, Sofia,

Bulgaria

H. Lochmuller

Institute of Genetic Medicine, Newcastle University,

Newcastle upon Tyne, UK

L. Kalaydjieva (&)

Western Australian Institute for Medical Research,

‘‘B’’ Block QE II Medical Centre, Hospital Ave,

Nedlands WA6009, Australia

e-mail: luba@waimr.uwa.edu.au

I. Tournev

Department of Cognitive Science and Psychology,

New Bulgarian University, Sofia, Bulgaria

123

J Neurol (2012) 259:906–911

DOI 10.1007/s00415-011-6276-6

and the currently known 14 genes do not provide complete

coverage for molecular diagnosis and prevention [4].

Recently, an AR form of ataxia due to mutations in the

Anactomin 10 gene (ANO10) was described in three fam-

ilies of diverse ethnic background [1]. One was a Roma/

Gypsy family from Serbia with three affected siblings

homozygous for a frameshift mutation in ANO10,

c.1150_1151del (p.Leu384fs). The clinical phenotype in

patients with ANO10 mutations was characterized by late

onset of the ataxia (varying widely between 13 and

45 years), brisk knee reflexes, gaze-evoked downbeat

nystagmus with hypermetric saccades, lower motor neuron

involvement, and severe cerebellar atrophy on neuroim-

aging. There was a degree of heterogeneity between the

three families [1] that could be related to the nature of the

mutations, with some distinctive features in the Gypsy

patients carrying the truncating defect.

The Roma/Gypsies are a founder population of rela-

tively limited genetic diversity, where numerous autosomal

recessive disorders are caused by ancestral mutations

shared between apparently unrelated individuals [5]. Such

groups of patients provide an opportunity for studying the

spectrum of clinical manifestations on the homogeneous

genetic background of a single mutation, avoiding the

complexity of genotype-phenotype correlations in outbred

populations [6]. The identification of an ANO10

c.1150_1151 deletion in a Gypsy family from Serbia with

ataxia [1] led us to hypothesise that it may be another

Gypsy founder mutation, with affected subjects likely to

exist in neighbouring countries. This study aimed at the

identification and phenotype characterization of such cases

and an assessment of ANO10 c.1150_1151del carrier rates

in a sample of population controls.

Subjects and methods

Subjects

We searched the archives and clinical notes accumulated

over 15 years of field work with the Roma/Gypsy popu-

lation of Bulgaria, as well as in-patient records of the

Neurology Department of the Medical University in Sofia,

for cases with an ataxia phenotype similar to the one

described in the original report [1].

A single family with three affected children, born to

healthy unrelated parents, came to our attention. A total of

10 family members from three generations participated in

the study (Fig. 1).

The panel of healthy controls (Supplementary Table 1)

included a total of 513 individuals of Roma/Gypsy ethnic-

ity, representing diverse sub-isolates [7, 8] and 13 subjects

of northwestern Indian origin residing in Western Australia.

Written informed consent was obtained from all par-

ticipants. The study complies with the ethics guidelines of

the institutions involved.

Clinical investigations

Phenotype assessment was based on the data collected

during admission to the clinic in 2006, brief hospitalization

during the present study and two home visits by the

research team. Information on early development and on

disease history was obtained from interviews with unaf-

fected family members.

Detailed neurological and ophthalmological examina-

tions were performed in the affected individuals. The

neurological assessment included a modified version of the

ataxia scale [9] evaluating dysarthria, dysdiadochokinesia,

dysmetria, gait and stance, each on a scale of 0–4 (4

equaling severe impairment). Nerve conduction studies

(NCS) and electromyography (EMG) using a Dantec–

Keypoint portable electromyograph (Natus, Copenhagen,

Denmark) complemented by concentric needle electromy-

ography (EMG) and standard techniques, were performed

in patients II.4 and II.5. Magnetic resonance imaging

(MRI) of the brain was performed in II.4 on a 1.5T MR

imager (MR Signa HDxt, GE Healthcare Milwaukee USA).

Formal neuropsychological testing was performed in the

three affected subjects, their two unaffected siblings and in

nine additional age-matched healthy controls of the same

ethnicity and educational background. We used a 2 h test

battery, comprising the Mini-Mental State Examination

(MMSE), general intelligence assessment using Raven’s

progressive matrices, memory tests including Rey’s Audi-

tory Verbal Learning Test (immediate and delayed word

recall and forced-choice recognition [10]) and digit span

forward, word fluency (phonological and semantic), and

executive function (Tower of London and digit span

backward). Performance was scored following the standard

procedures outlined in test manuals.

Genotyping

The c.1150_1151del mutation was analysed by sequencing

exon 6 of the ANO10 gene (primers: 50-GGGTCTGAGT

GGACCAGTGT-30 and 50-CAACATCTATTTTCTCTGC

AAGGT-30). The control panel was screened for the muta-

tion by high-resolution melting analysis (primers 50-TGT

TGTATGTGCCCAGCATC-30 and 50-TGTGAATCCCAT

GATCTAGGC-30), using the LightCycler 480 system and

the High Resolution Melting Master reagents (Roche

Diagnostics Pty Limited). The data were analysed with the

dedicated LightCycler� 480 software. Carrier status was

confirmed independently using Sanger sequencing.

J Neurol (2012) 259:906–911 907

123

Table 1 Clinical features of

patients with autosomal

recessive cerebellar ataxia

caused by the c.1150_1151

deletion in ANO10

* Based on a modified version

of the ataxia scale [9] evaluating

dysarthria, dysdiadochokinesia,

dysmetria, gait and stance, each

on a scale of 0–4 (with 4

equaling severe impairment)

Patient II.2 II.4 II.5

Year of birth 1975 1978 1981

Sex M F M

Early development Normal Normal Normal

Age at onset (years) Dysarthria: 6–7;

Ataxia: 16

Dysarthria: 6–7; Ataxia: 17 Dysarthria: 7;

Ataxia:17

Age at most recent

examination (years)

35 32 29

Ocular pursuit Horizontal and

vertical nystagmus

Downbeat nystagmus Downbeat

nystagmus

Cerebellar dysarthria Moderate Moderate Moderate

Dysmetria and

dysdiadochokinesia

of limbs

Mild Mild Mild

Gait ataxia Severe Moderate Moderate

Appendicular ataxia Mild Mild Mild

Ataxia score* 7/16 6/16 6/16

Tendon reflexes: UL Increased Increased Increased

Tendon reflexes: LL

(knee)

Increased Increased Increased

Tendon reflexes: LL

(ankle)

Normal Increased Normal

Plantar responses Normal Extensor Normal

Nerve conduction Not studied Normal Normal

EMG Not done Normal (2006), Motor neuron

involvement in m.quadriceps

femoris (2011)

Normal (2006

and 2011)

Brain MRI Not done Severe cerebellar atrophy Not done

Intellectual deficit Following

development of

ataxia

Following development of ataxia Following

development

of ataxia

Co-morbidity Gastric ulcer Syphillis, treated with

Benzylpenicillin

None reported

Fig. 1 Pedigree structure of the

Roma/Gypsy family with the

p.Leu384fs mutation. Only

family members participating in

the study are labelled.

Genotypes are shown as either

N/N (homozygous normal),

N/del (heterozygous) or del/del

(homozygous for the

c.1150_1151 deletion, causing

the p.Leu384fs mutation in

ANO10)

908 J Neurol (2012) 259:906–911

123

Results

Analysis of the c.1150_1151del mutation confirmed

homozygosity in the three affected siblings. The healthy

parents and four additional unaffected family members

were carriers (Fig. 1).

Phenotype characterization

The clinical findings are summarized in Table 1. Pregnancy,

delivery and the neonatal period were described as

uneventful in all three affected individuals. Early psycho-

motor development was normal: the children started walking

at age 12–14 months and formed simple sentences around

18 months. Individuals II.2 and II.4 had 4 and 2 years of

schooling, respectively, while II.5 never went to school.

Onset of symptoms was at age 6–7 years, when slight

speech impairment was first noted; the dysarthria pro-

gressed slowly in subsequent years. Gait ataxia developed

around age 16–17 years and by 28–29 years became so

prominent that walking without support was impossible.

Slowing in daily activities and memory problems became

apparent subsequent to the onset of gait disturbances.

Upon recent neurological examination, gait ataxia was

severe in the oldest patient II.2 (age 35 years) and mod-

erate in the younger siblings. Limb dysmetria and dysdi-

adochokinesia were mild in all. The speech was moderately

dysarthric. Gaze-evoked downbeat nystagmus with hyper-

metric saccades was observed in the younger patients and

horizontal and vertical nystagmus in the oldest one. Tendon

reflexes were brisk in all three patients. Pathological

extensor responses were present only in II.4.

EMG in patient II.4 detected neurogenic changes in

m.quadriceps femoris with spontaneous activity of rare

positive sharp waves and fibrillation potentials and some

long duration action potentials; tibialis anterior and

extensor digitorum brevis showed no changes. The EMG

results were normal in patient II.5.

Brain MRI (Fig. 2) in II.4 showed severe diffuse cere-

bellar atrophy and diffuse T2/FLAIR hyperintense and T1

Fig. 2 Brain MRI of patient

II.4, documenting severe

cerebellar atrophy with normal

supratentorial structures, as well

as diffuse hyperintense zones on

T2 and hypointense on T1 in the

cerebellum, consistent with

gliosis. 2A Sagittal T1, 2B Axial

T2 PROPELLER, 2C Coronal

T2 FLAIR

J Neurol (2012) 259:906–911 909

123

hypointense zones in the cerebellar hemispheres, consistent

with gliosis. The supratentorial structures were normal.

In the original description [1], tortuosity of conjunctival

vessels and mental retardation were listed as distinct phe-

notypic features in c.1150_1151del homozygotes. In our

patients, tortuosity of conjunctival vessels was not present;

however, ophthalmological examination revealed bilateral

macular hypoplasia and tortuosity of retinal vessels in

patient II.2.

Since language differences (many Gypsy families speak

Romanes at home), and cultural and educational back-

ground not infrequently lead to Gypsy individuals (espe-

cially children) with normal intellect being dubbed as

disabled, we undertook a formal assessment of cognitive

function in the three affected individuals. To compensate

for the lack of validated norms in the Gypsy population,

testing was also performed in the unaffected siblings and

in a small group of age- and ethnicity-matched controls.

The results of the affected subjects in comparison to both

their siblings and to unrelated controls (Table 2) revealed

wide-spread deficits across most cognitive domains.

The data do support moderate intellectual deficit in the

patients.

Mutation carrier rates

Screening of the 526 controls for the mutation (Supple-

mentary Table 1) detected a single heterozygote, a control

subject of Gypsy ethnicity from Romania, corresponding to

a carrier rate of 0.2% in the overall sample of European

Gypsies and 3% in the sample from Romania. No carriers

were found among northwestern Indians.

Discussion

Here we report a second Gypsy family with autosomal

recessive ataxia caused by the same truncating ANO10

defect as that described by Vermeer et al. [1], confirming

p.Leu384fs as another founder mutation in this population.

No further patients or families were identified during our

brief search, but their existence is suggested by the

ancestral origin of the mutation and by the lack of parental

consanguinity in our family. While our current data point

to a very low heterozygote frequency, this should be

interpreted in the context of the structured Gypsy popula-

tion. Carrier rates for a given mutation are modulated by

population history and genetic drift, and can vary dramat-

ically between sub-isolates [8]. Sub-isolates with higher

carrier rates may still exist but are not represented in our

control panel. The p.Leu384fs mutation should thus be

considered in the diagnostic testing of subjects of Gypsy

ethnicity who display a similar phenotype.

Our data outline a relatively homogeneous clinical

presentation in the six currently known ataxia patients

homozygous for the p.Leu384fs mutation, with a more

severe phenotype likely due to the complete Anactomin 10

deficit caused by the truncating mutation. The most con-

sistent features, supporting a more severe clinical course in

these patients, compared to the others described in [1],

Table 2 Neuropsychological testing of the affected subjects, unaffected siblings and unrelated healthy controls

Individuals Patients Unaffected siblings Healthy controls N = 9

mean values (SD)*II.2 II.4 II.5 II.1 II.3

Raven progressive matrices-raw scores 19/60 13/60 12/60 25/60 22/60 24.78 (6.0)

MMSE 21 19 21 27 26 24.56 (1.74)

Digit span

Forward 2 1 2 6 7 5.56 (1.13)

Backward 2 2 1 7 7 3.89 (1.05)

Verbal fluency

Semantic ‘‘animals’’ 5 4 4 10 12 10.11 (2.26)

Phonemic ‘‘M’’ 2 3 1 4 3 2.56 (1.13)

Rey verbal learning test

Immediate recall 23/75 24/75 13/75 36/75 36/75 36.78 (6.10)

Delayed recall 2/15 3/15 3/15 7/15 8/15 9.89 (1.83)

Recognition 20/30 20/30 12/30 27/30 29/30 27.56 (1.67)

Tower of London

Total move score 51 65 61 35 42 46.89 (9.58)

Total correct scores 1 0 0 3 2 2.00 (1.41)

* Analyzed by the non-parametric Mann–Whitney U test. Statistical analyses were conducted in SPSS for Windows, version 13.00

910 J Neurol (2012) 259:906–911

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include earlier onset and cognitive decline. The onset of

gait ataxia was between 13 and 17 years, compared to

20–45 years in the families with other ANO10 mutations,

while dysarthria (not specified in the previously reported

cases) already developed around age 6–7 years. The

intellectual disability present in the Serbian Gypsy patients

[1] was substantiated in our family. Cognitive testing

showed a generalized deficit, whose evolution (lack of

adverse perinatal events, normal early development and

gradual emergence of memory problems and declining

coping skills concomitant with the development and pro-

gression of other ataxia manifestations) indicates a

degenerative rather than a neurodevelopmental process.

These data support the increasingly recognized role of the

cerebellum in the regulation and coordination of cognitive,

as well as motor processes. The results are consistent with

previous studies, showing the important role of the cere-

bellum in attention [11, 12] and verbal working memory

[12] due to processing of information via frontal and

parietal corticocerebellar loops [13]. At the same time, one

should note the high ANO10 expression levels in brain

areas important for cognitive performance, such as the

cortex and hippocampus [1], suggesting that the encoded

protein may have multiple functions in need of further

investigations.

While the tortuosity of conjunctival vessels noted in the

original description [1] was not supported in our study, one

of our patients had retinal developmental abnormalities

which do not seem to be related to the ataxia phenotype but

may suggest a role of the gene in eye development.

Severe cerebellar atrophy without involvement of the

supratentorial structures seems to be generally typical of

ataxia patients with ANO10 mutations, whereas lower

motor neuron involvement and downbeat nystagmus are

common but not invariable features.

Acknowledgments We thank the family members and controls

participating in this study.

Conflict of interest The authors declare that they have no conflict

of interest.

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