Mutation screening of the DYT6/THAP1 gene in Italy 


Mutation Screening of the DYT6/
THAP1 Gene in Italy

Monica Bonetti, BSc,
1
Chiara Barzaghi, PhD,

2

Francesco Brancati, MD, PhD,
1,3

Alessandro Ferraris, MD, PhD,
1

Emanuele Bellacchio, PhD,
1

Alessandro Giovanetti, BSc,
2
Tamara Ialongo, MD, PhD,

4

Giovanna Zorzi, MD,
5
Carla Piano, MD,

4

Martina Petracca, MD,
4
Alberto Albanese, MD,

6

Nardo Nardocci, MD,
5
Bruno Dallapiccola, MD,

1

Anna Rita Bentivoglio, MD, PhD,
4

Barbara Garavaglia, PhD,
2

and Enza Maria Valente, MD, PhD
1,7*

1IRCCS Casa Sollievo della Sofferenza, CSS-Mendel Institute,
Rome, Italy; 2Unit of Molecular Neurogenetics, Department
of Child Neuropsychiatry and Department of Neurology,
IRCCS Neurologic Institute Carlo Besta, Milan, Italy;

3Department of Biomedical Sciences, Ce.S.I. Aging Research
Center, Gabriele d’Annunzio University Foundation,

Chieti, Italy; 4Institute of Neurology, Catholic University,
Rome, Italy; 5Department of Child Neuropsychiatry, IRCCS
Neurologic Institute Carlo Besta, Milan, Italy; 6Department.

of Neurology, IRCCS Neurologic Institute Carlo Besta,
Milan, Italy; 7Department of Medical and Surgical Pediatric

Sciences, University of Messina, Messina, Italy

Video

Abstract: Mutations in the THAP1 gene on chromosome
8p21-p22 (DYT6 locus) have been recently reported as
causative of autosomal dominant primary torsion dystonia
(PTD) in four Amish–Mennonite families and in 12 addi-
tional probands of different ancestry. We sequenced the
THAP1 gene in 158 patients with DYT1-negative PTD
who had onset of symptoms below 30 years and/or posi-
tive family history. One sporadic Greek male patient,
aged 57 years, was found to carry a novel heterozygous
missense variant in THAP1 exon 3 (p.Cys170Arg), of
likely pathogenic significance. This subject first presented
with right writer’s cramp at age of 10 years and, subse-
quently, developed left arm dystonia and an extremely
severe left laterocollis, without further spreading to other
body districts. Our findings expand the genotypic
spectrum of THAP1 and strengthen the association with

upper body involvement, including the cranial and cervi-
cal districts that are usually spared in DYT1-PTD. �
2009 Movement Disorder Society

Key words: primary torsion dystonia; DYT6; THAP1; tor-
ticollis

Primary torsion dystonia (PTD) is characterized by

sustained muscle contractions, causing twisting and re-

petitive movements and abnormal postures, and can be

inherited as an autosomal dominant or recessive trait.
1

Only two genes (TOR1A/DYT1 and THAP1/DYT6)
have been identified so far that cause autosomal domi-

nant PTD with reduced penetrance. A 3-base pair

(GAG) deletion in the TOR1A gene represents a com-
mon cause of early-onset dystonia, especially among

Ashkenazi Jews.
2

The DYT1 phenotype usually

presents with onset in a limb and rapid generalization,

with sparing of the cranial-cervical district.
2–4

Recently, mutations in the THAP1 (thanatos-associated
protein domain-containing apoptosis-associated protein

1) gene have been identified in DYT6-linked families,

including a founder Amish-Mennonite mutation.
5
Two

large screenings of PTD patients have subsequently

identified THAP1 mutations in 9 of 36 (25%)
DYT1-negative families with early-onset nonfocal PTD

and in 2 of 160 (1%) patients with PTD that was

early-onset and/or generalized and/or familial and/or

involving the face and laryngeal districts.
6,7

In the 50

patients with THAP1 mutations so far reported, the
DYT6 phenotype appears to be characterized by onset

mostly in the first two decades, with generalization in

about half cases and frequent involvement of the upper

body, including arms, cranial, and cervical districts. In

particular, up to 78% patients presented cranial dysto-

nia, of whom a large proportion experienced speech

problems related to dysarthria and/or spasmodic dys-

phonia.
6,7

Aim of this study was to investigate the frequency

and phenotypic spectrum of THAP1 mutations in a
large cohort of PTD patients ascertained in Italy.

PATIENTS AND METHODS

The THAP1 gene was first tested in 130 patients
with familial or sporadic early-onset PTD, recruited

from the movement disorders centers of the Catholic

University in Rome and the C. Besta Institute in Milan.

An arbitrary cut-off has been set at 30 years, as >90%
of known patients with THAP1 mutations had an onset
below this age.

5–7
We subsequently tested a second

group of 28 PTD probands with onset above 30 years

The first two authors contributed equally to this work.
Additional Supporting Information may be found in the online

version of this article.
*Correspondence to: Dr. Enza Maria Valente, Neurogenetics Unit,

CSS-Mendel Institute, viale Regina Margherita 261, Rome 00198,
Italy. E-mail: e.valente@css-mendel.it

Potential conflict of interest: None reported.
Received 17 July 2009; Revised 30 September 2009; Accepted 2

October 2009
Published online 11 November 2009 in Wiley InterScience

(www.interscience.wiley.com). DOI: 10.1002/mds.22861

2424 M. BONETTI ET AL.

Movement Disorders, Vol. 24, No. 16, 2009



and positive family history. All 158 patients tested

negative for the DYT1 deletion, fulfilled a diagnostic
flow-chart for dystonia

8
and had no signs of secondary

dystonia. Inclusion criteria did not consider either the

site of onset or the distribution of dystonia at latest ex-

amination (Table 1). The project was approved by the

local ethics committees and written informed consent

was obtained by all patients or legal representatives.

The three THAP1 exons and exon-intron junctions
were amplified from genomic DNA by polymerase

chain reaction (primers and conditions available upon

request), purified and sequenced bidirectionally using

the Big Dye Terminator chemistry and an ABI PRISM

3130XL automated sequencer (Applied Biosystems).

Multiple sequence alignments of the human THAP1

protein and its orthologues were generated by Clus-

talW software (http://www.ebi.ac.uk/clustalw/). Predic-

tion of the possible impact of the identified variant on

the protein function was obtained using PolyPhen

(http://genetics.bwh.harvard.edu/pph/) and SIFT (http://

sift.jcvi.org/) software. The structure of THAP1 in the

amino acid range 148–185 has been modeled with the

program MODELLER� 9v7, using as a template the

crystallographic structure of General control protein

GCN4 (PDB entry: 2efr).
9
Similarly, the putative dime-

rization between two THAP1 molecules has been

simulated by reproducing the same topology of interac-

tions observed for the dimer of GCN4.

RESULTS

Only 1 patient with early-onset PTD was found to

carry a novel heterozygous missense variant in THAP1

exon 3 (c.508T>C, p.Cys170Arg). This 57-year-old
sporadic male patient was born in Xanthi (in the

Northern Greek region of Thraki) from Greek ances-

tors; pregnancy, delivery, psychomotor development

were uneventful. At age 10, he presented with right

writer’s cramp. Over the following 6 years, he devel-

oped abnormal posturing of the left arm and neck. The

latest progressed in a few years to a severe left latero-

collis associated with levoscoliosis, much more inva-

lidating than the writer’s cramp (see video). Dystonia

failed to improve with anticholinergics, tetrabenazine,

and myorelaxants. Botulinum toxin injections improved

pain but failed to affect the dystonic posture. No other

muscular districts have become affected over time, and

dystonia remained segmental. In particular, no cranial

or speech involvement could be observed. Brain mag-

netic resonance imaging and routine testing were nor-

mal. His parents died at 75 and 97 years without mani-

festing any dystonic feature; as far as the patient

recalls, none of his family members (including his 60-

year-old brother and 23-year-old daughter) is affected

by any movement disorder.

The p.Cys170Arg variant affects an highly con-

served residue in the coiled coil domain (CCD) (aa

139–190), was predicted as damaging both by Poly-

Phen (PSIC score: 3.168) and SIFT (score: 0.03), and

it was not detected in 540 control chromosomes (Fig.

1). To gain insights into the effects of this mutation

on the protein, we generated a structural model of

THAP1 CCD (aa 148–185). We observed that all

hydrophobic amino acids faced the same side of the

CCD, while charged residues were regularly placed

outwards. This pattern of alternating charged and

hydrophobic residues in this specific region of

THAP1 is consistent with potential intermolecular

interactions between CCDs of two proteins. This is in

line with the results of a previously reported large

scale yeast-two-hybrid assay, that identified several

putative THAP1 interactors among which the THAP1

protein itself, suggesting homodimerization.
10

We

have therefore generated a possible model of self-

interaction between two THAP1 molecules, although

it is expected that THAP1 also heterodimerizes with

different proteins through the same CCD. In our

model, the Cys170 residue fell at the interface

between the two molecules and replacement of this

residue with an arginine produced a disruptive effect

on the predicted intermolecular interaction (Fig. 1). In

addition, the Cys170Arg change introduces a posi-

tively charged residue that likely interacts with the

negatively charged Glu174 forming an intramolecular

salt-bridge that perturbs THAP1 structure.

TABLE 1. Demographic and clinical data of tested patients

Early-onset PTD Late-onset PTD

Number (sex M/F) 130 (75M/55F) 28 (6M/22F)
Origin 128 Italian

a
All Italian

Age at onset 13.3 6 8.6 (1–29) 53.1 6 12.0 (32–73)
Age at examination 34.0 6 17.0 (1–75) 67.6 6 10.9 (45–80)
Family history
Positive 26 28
Negative 104 0

Distribution of dystonia
Focal 34 10
Segmental 31 18
Multifocal 3 0
Generalized 54 0
unknown

b
8 0

Ages at onset and at examination are presented as mean 6 stand-
ard deviation (range).

a
One Greek, one Albanian.
b
Latest examination <1 year from onset.

2425DYT6/THAP1 DYSTONIA IN ITALY

Movement Disorders, Vol. 24, No. 16, 2009



DISCUSSION

We detected a THAP1 missense variant of potential
pathogenic significance in 1 of 158 PTD patients

(0.6%), which is broadly in line with the 1% frequency

reported in a recent study on European patients.
7
These

figures are markedly lower than the 25% frequency

reported by Bressman et al who included in the screen-

ing only familial cases in which at least 1 patient had

nonfocal dystonia with onset below 22 years.
6
Yet, it

must be noted that both our and the 2 patients carrying

THAP1 mutations reported by Djarmati et al. were
sporadic, although one of these had two asymptomatic

relatives with subtle signs of dystonia who also carried

the mutation.
7

This lack of family history can be

explained by the reduced penetrance of THAP1 muta-
tions, although we were not able to confirm this in our

patient since both parents were dead and healthy rela-

tives were not available for genetic testing. Indeed, we

found no positive cases among our 17 probands who

matched the inclusion criteria of the Bressman study,
6

and the mutation frequency in our cohort reached only

1.3% among patients with nonfocal dystonia and onset

below 22 years (n 5 76). Moreover, the only subject

carrying a THAP1 missense change in our cohort was
from Northern Greece, whereas none of the 156 Italian

patients carried pathogenic mutations. Thus, despite

two families of Italian ancestry have been previously

reported bearing THAP1 mutations,6 it appears that
DYT6-dystonia represents a rare occurrence in Italy.

Our patient presented with segmental upper limb

and neck dystonia in the absence of any facial, lingual,

jaw or laryngeal involvement, supporting the conclu-

sion that laryngeal dystonia or speech involvement,

albeit frequently observed, are not mandatory features

of the DYT6 phenotype.
6,7

However, the presence of

cranial-cervical involvement may help differentiate this

form from DYT1-PTD, that could present similar fea-

tures but usually spares the cranial-cervical regions.
4,11

The THAP1 gene encodes a protein characterized by
a conserved putative DNA-binding motif at the N-ter-

minus, a proline-rich region, and a large coiled coil

region at the C-terminus, which includes a nuclear-

localization domain (Fig. 1).
5
Overall, eleven muta-

tions including six missense, one nonsense, and four

frameshift mutations have been described to date, with

no obvious genotype-phenotype correlations. The novel

FIG. 1. Characterization of the p.Cys170Arg mutation. A: Schematic of the protein encoded by THAP1. DBD, THAP DNA-binding domain; PR,
low-complexity proline-rich region; CCD, coiled-coil domain, including the nuclear localisation signal (dashed). B: Multiple alignment of THAP1
orthologues (aa 145–185). Hydrophobic residues are highlighted in grey, Cys170 is indicated by an arrow. C Model of homodimerization of
THAP1. Each monomer (aa 148–185) is represented as a ribbon. Hydrophobic residues and Cys170 are shown respectively as sticks and spheres
on each monomer.

Movement Disorders, Vol. 24, No. 16, 2009

2426 M. BONETTI ET AL.



missense change identified in our study (p.Cys170Arg)

is of likely pathogenic significance because it was not

detected in over 500 Caucasian control chromosomes

(although we did not have access to control samples

from Northern Greece), and it was predicted to be del-

eterious by two distinct bioinformatic prediction soft-

ware (PolyPhen and SIFT) and affected a highly con-

served amino acid across species. Interestingly, the

mutant residue represents the first variant to fall within

the CCD of the protein, since all other missense

changes clustered within the DNA binding domain.

Although the function of the CCD is still largely

unknown, it has been suggested that THAP1 may

homo- or heterodimerize with another THAP1 protein

or another member of the THAP family through this

domain, and our molecular modeling results would be

in favor of this hypothesis. These protein–protein inter-

actions may be critical to enhance the DNA binding

activity of the THAP1 zinc finger domain, which

appears to be relatively weak, and in this light a muta-

tion disrupting such intermolecular interactions may

severely hamper the key function of DNA binding.
12

In conclusion, our results expand the mutational

spectrum of DYT6-PTD and implicate that this genetic

form of dystonia is rare in Italy.

LEGEND TO THE VIDEO

The video illustrates the features of dystonia in the

57-year-old Greek patient carrier of the p.Cys170Arg

mutation in the THAP1 gene.
Segment 1. (Year 2004), Note the marked left latero-

collis with subsequent limitation in neck movements, and

the typical ‘‘geste antagoniste’’ (moving the right arm to

the head) that completely abolishes the dystonic posturing

of the neck. When the patient is sitting with his arms out-

stretched, there is dystonic posturing of both arms.

Segment 2. (Year 2009), The left laterocollis and

the arm dystonic posturing have not changed over

time. Note the writer’s cramp when the patient is writ-

ing and the levoscoliosis associated with neck dystonia

while the patient is walking.

Acknowledgments: This work was supported by the Ital-
ian Ministry of Health (Ricerca Corrente 2009; Ricerca Final-
izzata). The support of Fondazione Livio Patrizi and Transge-
nomics is also gratefully acknowledged. Part of DNA sam-
ples were obtained from the ‘‘Bank for the diagnosis and
research of Movement Disorders’’ (B.G.) of the EuroBioBank
network (www.eurobiobank.org).

Financial Disclosure: Alberto Albanese: occasional con-
sultancy advisory boards and honoraria from drug companies:
TEVA, Allergan, Merz, IPSEN; Anna Rita Bentivoglio: occa-

sional consultancy and honoraria from Allergan, IPSEN,
Novartis, Boheringer Ingheleim, UCB-Pharma. Monica
Bonetti, Chiara Barzaghi, Francesco Brancati, Alessandro
Ferraris, Emanuele Bellacchio, Alessandro Giovanetti, Tam-
ara Ialongo, Giovanna Zorzi, Carla Piano, Martina Petracca,
Nardo Nardocci, Bruno Dallapiccola, Barbara Garavaglia,
Enza Maria Valente have nothing to disclose concerning any
source of financial support and funding (including: stock
ownership in medically-related fields, consultancies, advisory
boards, partnerships, honoraria, grants, intellectual property
rights, expert testimony, employment, contracts, royalties,
etc) for the preceding 12 months, regardless of relationship
to this manuscript.

Author Roles: M. Bonetti and A. Ferraris were involved
in execution of the project, data analysis, and manuscript
writing; C. Barzaghi, F. Brancati, E. Bellacchio, and A. Gio-
vanetti, were involved in execution of the project and data
analysis; T. Ialongo, G. Zorzi, C. Piano, and M. Petracca
were involved in ascertainment of patients; A. Albanese, N.
Nardocci, B. Dallapiccola, and A.R. Bentivoglio were
involved in ascertainment of patients, review, and critique of
the manuscript; B. Garavaglia and E.M. Valente were
involved in conception and organization of the project,
review and critique of the manuscript

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Movement Disorders, Vol. 24, No. 16, 2009