Expansion of the first PolyA tract of ARX causes infantile spasms and status dystonicus

Article (PDF Available)inNeurology 69(5):427-33 · August 2007with70 Reads
DOI: 10.1212/01.wnl.0000266594.16202.c1 · Source: PubMed
Abstract
ARX is a paired-type homeobox gene located on the X chromosome that contains five exons with four polyalanine (PolyA) tracts, a homeodomain, and a conserved C-terminal aristaless domain. Studies in humans have demonstrated remarkable pleiotropy: malformation phenotypes are associated with protein truncation mutations and missense mutations in the homeobox; nonmalformation phenotypes, including X-linked infantile spasms (ISS), are associated with missense mutations outside of the homeobox and expansion of the PolyA tracts. To investigate the role of ARX, we performed mutation analysis in 115 boys with cryptogenic ISS. This included two pairs of brothers. We found an expansion of the trinucleotide repeat that codes for the first PolyA tract from 10 to 17 GCG repeats (c.333_334ins[GCG]7) in six boys (5.2%) ages 2 to 14, from four families, including the two pairs of brothers. In addition to ISS, all six boys had severe mental retardation and generalized dystonia that appeared around the age of 6 months and worsened, eventually leading to stable severe quadriplegic dyskinesia within age 2 years. Three children experienced recurrent, life-threatening status dystonicus. In four children brain MRI showed multiple small foci of abnormal cavitation on T1 and increased signal intensity on T2 in the putamina, possibly reflecting progressive multifocal loss of tissue. The phenotype of infantile spasms with severe dyskinetic quadriparesis increases the number of human disorders that result from the pathologic expansion of single alanine repeats. ARX gene testing should be considered in boys with infantile spasms and dyskinetic cerebral palsy in the absence of a consistent perinatal history.

Figures

Expansion of the first PolyA tract of
ARX causes infantile spasms and
status dystonicus
R. Guerrini, MD
F. Moro, PhD
M. Kato, MD, PhD
A.J. Barkovich, MD
T. Shiihara, MD
M.A. McShane, MD
J. Hurst, MD
M. Loi, MD
J. Tohyama, MD
V. Norci, BS
K. Hayasaka, MD
U.J. Kang, MD, PhD
S. Das, PhD
W.B. Dobyns, MD
ABSTRACT Background: ARX is a paired-type homeobox gene located on the X chromosome that
contains five exons with four polyalanine (PolyA) tracts, a homeodomain, and a conserved
C-terminal aristaless domain. Studies in humans have demonstrated remarkable pleiotropy: mal-
formation phenotypes are associated with protein truncation mutations and missense mutations
in the homeobox; nonmalformation phenotypes, including X-linked infantile spasms (ISS), are as-
sociated with missense mutations outside of the homeobox and expansion of the PolyA tracts.
Objective: To investigate the role of ARX, we performed mutation analysis in 115 boys with
cryptogenic ISS. This included two pairs of brothers.
Results: We found an expansion of the trinu-
cleotide repeat that codes for the first PolyA tract from 10 to 17 GCG repeats (c.333_334ins[GCG]7)
in six boys (5.2%) ages 2 to 14, from four families, including the two pairs of brothers. In addition to
ISS, all six boys had severe mental retardation and generalized dystonia that appeared around the age
of 6 months and worsened, eventually leading to stable severe quadriplegic dyskinesia within age 2
years. Three children experienced recurrent, life-threatening status dystonicus. In four children brain
MRI showed multiple small foci of abnormal cavitation on T1 and increased signal intensity on T2 in
the putamina, possibly reflecting progressive multifocal loss of tissue.
Conclusion: The phenotype of
infantile spasms with severe dyskinetic quadriparesis increases the number of human disorders
that result from the pathologic expansion of single alanine repeats. ARX gene testing should be
considered in boys with infantile spasms and dyskinetic cerebral palsy in the absence of a consis-
tent perinatal history.
NEUROLOGY 2007;69:427–433
ARX (aristaless-related homeobox) is a paired-class homeobox gene comprising five ex-
ons that contains highly conserved octapeptide, homeobox, and C-terminal domains as
well as four PolyA tracts.
1
It is expressed in embryonic brain, endocrine pancreas, testes,
and probably other tissues as well as in adult brain, heart, skeletal muscle, and liver.
1-4
Studies in humans have demonstrated remarkable pleiotropy, as several apparently unre-
lated disease phenotypes have been associated with mutations of this gene, including
syndromes with and without brain malformations. The former include X-linked lissen-
cephaly with abnormal genitalia (XLAG), severe hydrocephalus, and Proud syndrome
(agenesis of the corpus callosum with abnormal genitalia), whereas the latter include
X-linked infantile spasms (ISS), Partington syndrome, which consists of mental retarda-
tion with mild distal dystonia, and nonspecific X-linked mental retardation.
3,5-10
In gen-
eral, the malformation phenotypes are associated with protein truncation mutations and
missense mutations in the homeobox, whereas the nonmalformation phenotypes are as-
sociated with missense mutations outside of the homeobox and expansion of the PolyA
tracts.
While investigating the role of ARX as the causative gene for cryptogenic infantile
spasms, we found an expansion of the trinucleotide repeat that codes for the first PolyA
From the Department of Pediatric Neuroscience (R.G., V.N.), Pediatric Hospital A. Meyer and University of Florence, Research Institute
I.R.C.C.S. Stella Maris Foundation (R.G., F.M.), Pisa, and Azienda Ospedaliera G. Brotzu (M.L.), Servizio di Neuropsichiatria Infantile,
Cagliari, Italy; Department of Pediatrics (M.K., T.S., J.T., K.H.), Yamagata University School of Medicine, Japan; Department of Radiology
(A.J.B.), University of California, San Francisco, and Departments of Neurology and Neurobiology, Pharmacology, and Physiology (U.J.K.)
and Human Genetics (S.D., W.B.D.), University of Chicago, IL; and Paediatric Neurology (M.A.McS.), Oxford Radcliffe Hospitals, and
Department of Clinical Genetics (J.H.), Churchill Hospital, Oxford, UK.
Funded partly by the Italian League Against Epilepsy (to R. Guerrini) and by a collaborative study group for West syndrome in Japan and
supported by a grant from the Ministry of Education, Science, Sports, and Culture of Japan and by a grant from the Japan Epilepsy Research
Foundation (to M. Kato).
Disclosure: The authors report no conflicts of interest.
Supplemental data at
www.neurology.org
Address correspondence and
reprint requests to Dr. R.
Guerrini, Clinical Pediatric
Neurology, Department of
Pediatric Neurosciences,
University of Florence, and
Pediatric Hospital A Meyer,
via Bonvicini 62,
50132 Firenze- Italy
r.guerrini@meyer.it
Editorial, see page 421
ARTICLES
Copyright © 2007 by AAN Enterprises, Inc. 427
at UNIVERSITA DI FIRENZE on August 1, 2007 www.neurology.orgDownloaded from
tract in the protein from 10 to 17 GCG re-
peats (c.333_334ins[GCG]7) in six boys
from four families. All shared a severe clin-
ical picture of early infantile spasms with
generalized dystonia that appeared around
age 6 months and worsened, eventually
leading to severe quadriplegic dyskinesia
within age 2 years, accompanied in three
by relentless episodes of devastating gener-
alized dystonia and rigidity (status
dystonicus).
11
METHODS Patients. To investigate the role of ARX mu-
tations in cryptogenic ISS, we performed mutation analysis
in 115 boys (68 from Pisa, 31 from Yamagata, and 16 from
Chicago) with ISS and developmental delay, all of whom had
normal brain imaging and no recognized etiology. This in-
cluded two pairs of brothers. We defined ISS as an electro-
clinical syndrome of flexor, extensor, or flexor– extensor
spasms, appearing in clusters and accompanied on the EEG
by a bilateral electrodecremental event or biphasic slow
wave with superimposed low voltage fast rhythms, with or
without interictal hypsarrhythmia.
12
We found a (GCG)
10 7
PolyA expansion of the ARX gene in six boys (5.2%). One-
third of the patients, including all six described here, were
clinically examined; for the remaining patients, clinical in-
formation was obtained from the referring physicians
through a standard questionnaire. For all six boys, informed
parental consent was obtained for videotaping.
After noticing that all six boys carrying the (GCG)
10 7
PolyA expansion had severe dystonia and that three of them
had experienced status dystonicus, we decided to investigate
whether ARX mutations might be a frequent cause of status
dystonicus. However, we found no ARX gene mutations in
seven additional boys who had had status dystonicus of un-
known etiology that had not been preceded by ISS.
DNA extraction, PCR amplification, and mutation
analysis.
Blood or DNA samples were obtained from all
patients after informed parental consent. Genomic DNA
was extracted from blood samples using standard proto-
cols.
13
PCR amplification was performed with 50 ng of
genomic DNA template using Taq Gold DNA polymerase
(Applied Biosystems). The primers used to amplify the cod-
ing and flanking noncoding regions of ARX (GenBank no.
AY038071.1) were designed using Primer 3 software (http://
www-genome.wi.mit.edu/cgi-bin/primer/
primer3_www.cgi). PCR primer sequences are described in
table E-1 on the Neurology Web site (www.neurology.org).
Because of the high GC content of exon 2, we optimized the
amplification conditions using KOD-Plus-DNA polymerase
(TOYOBO-Novagen). PCR products were analyzed using
denaturing high performance liquid chromatography (DH-
PLC), followed by direct sequencing of PCR fragments that
showed abnormal DHPLC profiles on automated capillary
sequencer (ABI Prism 310; Applied Biosystems). DHPLC
conditions were chosen according to the Wavemaker pro-
gram (Transgenomic). With the exception of exon 2, primers
used for PCR and sequencing were the same.
Clinical reports. Patient 1. This 15-year-old boy from a
Sardinian family was born after a normal pregnancy with
birth head circumference 34 cm (50th percentile) (figure 1,
AIII:1). Two maternal uncles and two aunts who had died
between the 16th and 20th months of life were reported to
have severe developmental delay and intractable seizures.
Delayed milestones were noticed in the first months of
life. He had onset of ISS with hypsarrhythmia at 3 months,
which proved refractory to treatment. The spasms ceased by
8 months, but generalized dystonia and chorea appeared and
progressed in severity until 2 years. Exam demonstrated in-
creased tone and brisk tendon reflexes. He began to have
prolonged episodes of status dystonicus lasting days at a
time, with worsening of his dyskinesia, dyspnea, and inabil-
ity to swallow that prompted frequent hospital admissions.
These were treated with anticholinergic drugs with no bene-
fit and with apomorphine and benzodiazepines with limited
benefit. He also had infrequent focal seizures. EEG showed
multifocal epileptiform abnormalities. Brain MRI at age 10
Figure 1 Pedigrees, schematic representation of genomic structure, and
electropherograms
(A and B) Pedigrees of the Italian and Japanese families. A-III:1 Patient 1; A-III:2 Patient
2; B-II:1 Patient 5; B-II:2 Patient 6. Squares males; circles females, arrows
probands; filled black symbols patients with infantile spasms and dyskinetic quadriparesis;
right side quarter-filled symbols early seizures and severe neurologic impairment; dotted
circles carriers of ARX mutation; white symbols unaffected individuals; dashed
symbols deceased individuals. (C) Schematic representation of the genomic structure of
ARX and DNA sequence electropherograms showing the (c.333_334ins[GCG]7) mutation
located in the first PolyA tract. The electropherograms show the sequence of the first
PolyA tract (black lines) in a wild-type (WT) individual and the sequence with the expansion
(GCG)
10 7
(black arrow) in the probands. This mutation introduces seven extra alanine (A)
residues in the ARX protein.
428 Neurology 69 July 31, 2007
at UNIVERSITA DI FIRENZE on August 1, 2007 www.neurology.orgDownloaded from
showed multiple small areas of high T2 signal in the poste-
rior inferior regions of both basal ganglia that appeared to be
of CSF intensity on T1-weighted spin echo and inversion re-
covery (IR) sequences, as well as mildly enlarged ventricles.
Biochemical screening for a wide range of metabolic disor-
ders and muscle biopsy were normal. At age 15, he had mild
microcephaly (–2 SD), severe mental retardation with absent
speech, and severe dystonia (see video 1 on the Neurology
Web site). He was assigned the diagnostic label of “dyski-
netic cerebral palsy.” Brain MRI in his mother and maternal
aunt were normal.
Patient 2. The 9-year-old brother of Patient 1 had a sim-
ilar clinical history with onset of infantile spasms at 3
months and onset of generalized dystonia and chorea at 5
months that progressed in severity until 2 years (figure 1,
AIII:2). Recurrent status dystonicus was also observed. Brain
MRI at 5 years demonstrated multiple small cystic areas in
the basal ganglia as well as mildly enlarged ventricles. At age
9 he had continuous severe dystonia (see video 2). His head
circumference was 47 cm (50th percentile) at 1 year and 52
cm (25th to 50th percentile) at 9 years.
Patient 3. This 8-year-old British boy was first brought
to medical attention at 5 months with motor delay, fisting,
and ISS. Family and perinatal history were negative. He was
born at term with normal head circumference. By 1 year,
dystonic posturing was noted that progressed to generalized
dyskinesia over the following months, and a tentative diag-
nosis of “dyskinetic cerebral palsy” was assigned. At 3 years,
he experienced his first episode of status dystonicus, lasting
several days. Baclofen, benxhezol, benzodiazepines, and tet-
rabenazine were of little benefit. Focal seizures appeared at
this time and were treated with valproate and subsequently
carbamazepine monotherapy, which slightly improved the
involuntary movements. Brain MRI at 6 years showed small
areas of T1 hyperintensity in the mesencephalon (figure 2A)
as well as multiple small areas of high T2 signal in both basal
ganglia (not shown) that appeared to be small cysts on T1
and IR sequences (figure 2A) and mild ventricular enlarge-
ment. The nature of these is uncertain, but possibilities in-
clude foci of calcification, lipid, and proteinaceous fluid.
After 4 years of clinical stability, involuntary movements
evolved again into status dystonicus (see video 3) with hyper-
thermia, acidosis, hypernatremia, uremia, thrombocytope-
nia, coagulopathy, and elevated creatinine and creatine
kinase levels (52,590 U/dL). Treatment with haloperidol and
tetrabenazine probably triggered malignant neuroleptic syn-
drome, with life-threatening acidosis, disseminated intravas-
cular coagulation, and renal failure. His course remained
stormy with recurrent episodes of status dystonicus. Treat-
ment with carbamazepine, baclofen, diazepam, morphine,
nasal midazolam, and deep brain stimulation were of limited
benefit. Head circumference at birth and afterward was al-
ways at the 50th centile.
Patient 4. This 14-year-old boy has severe mental retar-
dation, dyskinetic movements, and intractable epilepsy.
Family history is negative. He was born at term with normal
head circumference. Perinatal history was unrevealing. He
had the onset of ISS at 2 months when an EEG showed mul-
tifocal spike or polyspike and wave activity, followed by
electrodecremental responses. He was started on adrenocor-
ticotropic hormone (ACTH) in addition to phenobarbital
and clonazepam. Spasms were transiently controlled but re-
lapsed at 6 months after stopping ACTH, and he was treated
with phenytoin. Over the next several months, the ISS finally
stopped, and his EEG improved; however, he went on to
have drug-resistant focal seizures and since age 12 he has had
multiple seizure types per day. During infancy and early
childhood, continuous choreoathetotic and dystonic move-
ments developed. He learned to walk on his knees, but al-
most constant twisting or writhing movements largely
prevent normal hand use (see video 4). MRI scan at 5 years
showed multiple small areas of high T2 signal in both basal
ganglia that appear to be small cysts based on T1-weighted
spin echo and IR sequences, as well as mildly enlarged ven-
tricles (figure 2B). His head circumference was 54.6 cm (50th
percentile) at 14 years.
Patient 5. This 3-year 6-month-old Japanese boy was
born at term as the first child of healthy and unrelated par-
ents (figure 1B, II:1). Head circumference at birth was 33.0
cm (about 10th percentile). His early development was
mildly delayed with visual tracking and smiling noted at 2
months, but he never acquired good head control. He had
the onset of brief clonic seizures at 3 months, followed by ISS
several weeks later that were associated with loss of develop-
mental milestones. An EEG showed hypsarrhythmia,
whereas brain MRI at 4 months was normal. After treatment
with several antiepileptic drugs was ineffective, ACTH con-
trolled the spasms at age 8 months, but they relapsed at 11
months, at which time generalized dystonic movements first
appeared. A second cycle of ACTH wa