Coronary stenosis mimicking epilepsy 583
graphy is the best method for diagnosis of coronary anomalies;
Fig. 4 Right coronary angiogram obtained on the right anterior
oblique position. Left coronary system is filled up totally by right
coronary artery retrogradely up to the left main coronary ostium.
seizures. Musiani et al.3 showed among 15 pediatric patients
suffering from LMCA atresia and stenosis that the main refer-
ral signs were myocardial infarction in younger children
and syncope and tachyarrhythmias in older children. The angio-
A case of d-bifunctional protein deficiency: Clinical, biochemical and
molecular investigations
Paolo Ghirri,1 Marco Vuerich,1 Sacha Ferdinandusse,4 Hans R. Waterham,2 Andrea Guzzetta,3 Maria C. Bianchi,2
Antonio Boldrini1 and Ronald J.A. Wanders4
1
Neonatology Unit, 2Department of Neuroradiology, S. Chiara Hospital, AOUP, and 3Department of Neurodevelopmental
Neuroscience, Stella Maris Scientific Institute, Pisa, Italy, and 4Lab Genetic Metabolic Diseases, Departments of Pediatrics
and Clinical Chemistry, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands
Key words d-bifunctional protein, peroxisomal fatty acid oxidation disorders, very long-chain fatty acids.
Correspondence: Paolo Ghirri, PhD, MD, U.O. di Neonatologia,
Ospedale S. Chiara, AOUP, via Roma 67, 56126 Pisa, Italy. Email:
pghirri@med.unipi.it
Received 19 May 2009; revised 16 June 2010; accepted 2 August
2010.
doi: 10.1111/j.1442-200X.2010.03255.x
coronary CT angiography can provide images of coronary mor-
phology and cardiac functions non-invasively.8 However, treat-
ment must be surgical with the aim of revascularization of
myocardium soon after the diagnosis.3
In conclusion, in cases presenting with syncope and seizures
related to effort, cardiac etiologies definitely must be taken into
account and myocardial stress tests should be performed even if
the physical examination, electrocardiogram, Holter electrocar-
diogram and echocardiography are normal and coronary artery
imaging must be performed, primarily in a non-invasive manner,
on unclear cases.
References
1 Frommelt PC, Frommelt MA. Congenital coronary artery anoma-
lies. Pediatr. Clin. North Am. 2004; 51: 1273–88.
2 Gebauer R, Cerny S, Vojtovic P, Tax P. Congenital atresia of the left
coronary artery-myocardial revascularization in two children. Inter-
act. Cardiovasc. Thorac. Surg. 2008; 7: 1174–5.
3 Musiani A, Cernigliaro C, Sansa M, Maselli D, De Gasperis C. Left
main coronary artery atresia: Literature review and therapeutical
considerations. Eur. J. Cardiothorac. Surg. 1997; 11: 505–14.
4 Goetz CG, Pappert EJ, eds. Textbook of Clinical Neurology, 1st edn.
WB Saunders, Philadelphia, PA, 1999.
5 Walczak TS, Leppik IE, Amelio MD et al. Incidence and risk factors
in sudden unexpected death in epilepsy: A prospective cohort study.
Neurology 2001; 56: 519–25.
6 O’Regan ME, Brown JK. Abnormalities in cardiac and respiratory
function observed during seizures in childhood. Dev. Med. Child.
Neurol. 2005; 47: 4–9.
7 Nei M, Ho RT, Sperling MR. ECG abnormalities during partial
seizures in refractory epilepsy. Epilepsia 2000; 41: 542–8.
8 Friedman AH, Fogel MA, Stephens P Jr et al. Identification,
imaging, functional assessment and management of congenital coro-
nary arterial abnormalities in children. Cardiol. Young 2007; 17:
56–67.
ped_3255 583..607
d-bifunctional protein (DBP), also called multifunctional protein
2, is a crucial component of the peroxisomal fatty acid beta-
oxidation system, and is required for the oxidation of multiple
fatty acids and fatty acid derivatives. It is a 79 kDa protein,
harboring two different enzymatic activities, a 2-enoyl-coenzyme
© 2011 The Authors
Pediatrics International © 2011 Japan Pediatric Society
584 P Ghirri et al.

A (CoA) hydratase activity, and a 3-hydroxyacyl-CoA dehydro-

genase activity. In addition to these two enzyme activities the
protein also contains a sterol-carrier protein-2-like unit.1–3 It has
been shown that DBP is indispensable for the breakdown of very
long-chain fatty acids, a-methyl-branched-chain fatty acids and
precursors of bile acids.4,5 DBP deficiency (MIM #261515) is an
autosomal recessive inborn error with a usually severe clinical
presentation, and most affected children die within the first two
years of life.6 The gene coding for human DBP (HSD17B4) was
mapped to chromosome 5q23.1, interval D59471-D593937. The
clinical picture is usually characterized by neonatal hypotonia,
seizures, psychomotor delay, craniofacial dysmorphic features,
neuronal migration defects and/or demyelination. We report an
infant with a clinical presentation suspicious for DBP deficiency,
which was subsequently confirmed by biochemical and molecu-
lar investigations.

Case report
The patient was a female, second child of a non-consanguineous
couple. The mother was gravida 3, para 2 with a previous twin
pregnancy interrupted at six months gestation due to fetal death
of one fetus and microcephaly of the other. Familial history was
unremarkable. There was no exposure to known teratogens
during pregnancy. Amniocentesis showed a normal female karyo-
type 46XX. Gravidic history was unremarkable until 36
weeks gestation, when fetal tachycardia was detected. Since the
tachycardia persisted, cesarean section was performed. Apgar
scores were 6 at 1 and 8 at 5 min. The infant’s birth weight was
2650 g (-1.46 SD), birth length was 49 cm (-0.11 SD), and
occipital-frontal circumference (OFC) was 34.5 cm (-0.13 SD).
Physical examination revealed craniofacial dysmorphism,
including a high forehead, low nasal bridge, high arched palate,
micrognathia, short neck with pterygium, funnel breast and short Fig. 1 High forehead, low nasal bridge, pterygium, funnel breast,
generalized hypotonia.
toes (Figs 1,2). She had severe generalized hypotonia, an almost
complete absence of reactivity and motility, absence of archaic
reflexes, and inconstant gaze. She was put on nasal continuous of fatty acids, and a significant signal reduction of the neurotrans-
airway pressure due to respiratory distress for 3 days. At 5 days mitter N-acetylaspartate (NAA) (Fig. 6). Brainstem auditory and
of life, she presented right-sided tonic-clonic seizures, which visually evoked responses showed delayed latencies on both
were treated with phenobarbital. Her first cerebral ultrasound sides.
examination at 2 days of life was normal. Cranial tomography
performed at 5 days of age did not reveal significant pathological
abnormalities. Later ultrasound controls revealed bilateral sub-
ependymal hemorrhages at 10 days of life, and cystic lesions and
enlargement of pericerebral spaces at 27 days of life. Brain mag-
netic resonance imaging (MRI) performed at 1 month of life
revealed malformation of the cortical mantle of the cerebral
hemispheres characterized by incomplete operculated sylvian
valley and a wide cortical dysplastic area with polymicrogyria
that involved the insular cortex and the frontotemporal opercula
of sylvian cisternae bilaterally. The dysplasia also involved the
cortex of the pre- and post-Rolandic gyri bilaterally (Fig. 3).
Delayed myelination, mainly involving the arcuate fibers of the
pre- and postcentral gyri, the corticospinal tracts and the deep
cerebellar white matter, was found (Figs 4,5). In vivo proton
magnetic resonance spectroscopy (1H-MRS) of interhemispheric
frontoparietal cortex highlighted an increased peak of lactate and Fig. 2 Micrognathia, short neck.

© 2011 The Authors

Pediatrics International © 2011 Japan Pediatric Society

Fig. 3 Diffuse cortical dysplasia of polymicrogyric aspect mainly
involving the insular and opercular cortex.
Electroencephalography showed poorly differentiated back-
ground activity with a significant excess of slow waves and
presence of frequent sharp elements; during the recording period
paroxysmal discharges of multifocal origin were recorded with
associated clinical features, such as tonic contraction of all four
limbs and left buccal deviation with homolateral closure of palpe-
bral fissure. At 2 months of life global hypokinesia and general-
ized hypotonia worsened; diphantoine and vigabatrin were added
to the phenobarbital to control increasing epileptic activity
mainly characterized by tonic-clonic episodes with shift to the
left of the head, left nystagmus, left arm extension, myoclonus of
the mouth and the tongue, and chewing episodes with sialorrhea;
sometimes the crises occurred in clusters of long duration, with
Fig. 4 Delay of myelination of the cerebellar white matter.
A severe case of DBP deficiency 585
Fig. 5 Delay of myelination of the pyramidal tract into the corona
radiata.
episodes occurring every 3–5 min. The infant did not achieve any
significant motor milestones and exhibited failure to thrive.
Opthalmological investigations initially showed mild left nuclear
cataract that subsequently worsened into severe bilateral catar-
act. Electrocardiogram, echocardiogram, total body X-ray, and
abdominal and renal ultrasonography were normal. Laboratory
studies including neonatal screening for inborn errors of metabo-
lism, TORCH serology, blood sugar, lactate, pyruvate, ammonia,
thyroid hormones, cortisol, and transaminases were normal. At 7
months she exhibited weight retardation (6500 g, -1.68 SD),
while length and OFC were normal for the age (70 cm, +0.86 SD;
46 cm, +2.03 SD, respectively). The patient died of heart failure
at 8 months of life. Post-mortem examination was not authorized
by the parents.
Fig. 6 In vivo proton magnetic resonance spectroscopy demon-
strates accumulation of lactate (Lac) and fatty acids (Lip) with a
decrease of N-acetyl-aspartate (NAA).
© 2011 The Authors
Pediatrics International © 2011 Japan Pediatric Society
586 P Ghirri et al.
Biochemical and molecular investigations
Plasma levels of very long fatty acids (VLCFA) at 22 days of life
were high: cerotic acid (C26:0) was 17.27 mmol/L (control range
0.42–1.0 mmol/L); C24:0/C22:0 was 2.12 (control range 0.95–
1.1); and C26:0/C22:0 was 0.91 (control range 0.019–0.06).
Because of these biochemical values and the clinical picture,
analysis of peroxisomal functions was performed in cultured
fibroblasts at the Laboratory of Genetic Metabolic Diseases of
the University Hospital Amsterdam when the child was 77 days
old. These investigations revealed normal activity of the peroxi-
somal enzyme dihydroxyacetone phosphate acyltransferase. Fur-
thermore, a clearly abnormal VLFCA profile was observed in the
fibroblasts: C24:0/C22:0 ratio was 2.85, control range (5%–95%)
1.55–2.30; C26:0/C22:0 ratio was 0.72, control range (5%–95%)
0.03–0.07; a decreased rate of b-oxidation of C26:0 (182 pmol/h
per mg protein, controls 1300 1 475); and a decreased rate of
b-oxidation of pristanic acid (7 pmol/h per mg protein, controls
1145 1 356). A normal rate of phytanic acid a-oxidation was
found. Normal immunoblot profiles for both acyl-CoA oxidase as
well as peroxisomal thiolase were found. Immunofluorescence
microscopy using antibodies raised against catalase, a peroxiso-
mal matrix protein, revealed the presence of peroxisomes,
although they were reduced in number and increased in size. All
these findings were compatible with an isolated defect in the
peroxisomal b-oxidation system. This conclusion was substanti-
ated by immunoblot analysis that revealed the absence of the 79
and 45 kDa bands of DBP with trace amounts of the 35 kDa
component. Molecular analyses revealed two splice site muta-
tions, IVS5-1G>T and IVS11+1Gdel, both in heterozygous form.
Subsequent analysis of DBP cDNA confirmed that the mutations
result in skipping of exon 6 and exon 11, respectively. Subse-
quently, molecular analysis of the DBP gene in the DNA of the
parents and sister of the proband was performed. The mother was
heterozygous for the IVS11+1Gdel mutation in the DBP gene,
while the father and the sister were both carriers of the IVS5-
1G>T mutation in the DBP gene.
Discussion
Our patient presented typical clinical abnormalities as described
for DBP deficiency, including neonatal hypotonia and seizures,
external dysmorphia, psychomotor delay, neuronal migration
defects and hypomyelination. She did not present liver disease
(jaundice past neonatal period, hepatomegaly). Importantly, she
showed a severe form of bilateral cataract, an uncommon feature.
During her hospital stay she did not achieve any significant
psychomotor milestones. In our patient, abnormal plasma
VLCFA levels were found as in most of the patients studied by
Ferdinandusse.6 In particular there is concordance between high
values of C24:0/C22:0 and C26:0/C22:0 ratios and C26:0 as
assessed in plasma and fibroblasts. Levels of the bile acid inter-
mediates di- and trihydroxycholestanoic acid have not been
assessed. As true for all DBP-deficient patients of the large cohort
study (126 patients),6 our patient had normal plasmalogen levels
in erythrocytes. Brain imaging in our patient revealed perisylvian
and rolandic polymicrogyria. Neocortical dysplasia (polymicro-
© 2011 The Authors
Pediatrics International © 2011 Japan Pediatric Society
gyria) is a common finding in DBP-deficient patients6 and it has
been found in 27 and 64% of patients at brain imaging and at
microscopy after brain autopsy, respectively. In the cohort of
Ferdinandusse demyelination of the cerebral and cerebellar
hemispheres occurred in 17 and 7% of the patients, respectively;6
myelination is the most important process of brain maturation. It
is a dynamic process that starts during fetal life and proceeds
predominantly after birth, until the end of the third year, in a
predefined and predetermined manner. In a normal brain during
the first month after birth myelination proceeds rapidly and on
MRI myelin becomes visible in the cerebellar white matter, cer-
ebellar peduncles, medial lemniscus and fasciculus longitudinalis
medialis, in the pons and mesencephalon, in the posterior limb of
the internal capsule and in the tracts from and to the precentral
and postcentral gyri in the corona radiata.8 In our patient MRI
performed at 29 days of age revealed a marked delay of myeli-
nation of the cerebellar white matter and of the pyramidal tract
into the corona radiata; no involvement of thalamus and globus
pallidus was found. 1H-MRS revealed in our patient a peak of
lactate and of fatty acids and a significant reduction of NAA; to
our knowledge, this is the first report on in vivo 1H-MRS data of
patients suffering from DBP. NAA is an amino acid uniquely
localized in neurons; therefore, this peak is considered a neuronal
marker and any cause of neuronal malfunction or rarefaction
turns into a NAA signal reduction. In our patient NAA levels, if
quantified relatively as ratios to the creatine (Cr) peak, showed a
NAA/Cr ratio of 1.1, while the normal range for the age is about
1.6–2.8.9 Total Cr is of approximately constant concentration in
the brain in different metabolic conditions; as such, the total Cr
concentration would be suitable as a standard for calculation of
ratios.9 Lactate is the end product of anaerobic glycolysis and it is
MRS detectable when brain oxidative metabolism is impaired
and the rate of this pathway is increased (like in the event of
hypoxia-ischemia and in many metabolic diseases). Fatty acids
signals come from abnormal myelin synthesis or from processes
of membrane disruption. In our patient some ultrasound findings
(subependimal hemorrhages and enlargement of pericerebral
spaces) were not confirmed by MRI studies. Our patient showed
severe bilateral cataract that is not a common finding in DBP
patients. In the cohort studied by Ferdinandusse, although no
specific questions were formulated in the questionnaire sent to
the referring physician of each patient, cataract was reported only
in four patients.6 As a consequence of the fact that d-bifunctional
protein is an enzyme composed of three functional units, DBP
deficiency can be divided into three subcategories: type I is a total
deficiency of both the hydratase and dehydrogenase activities of
DBP; type II is an isolated deficiency of the hydratase activity;
whereas type III is an isolated deficiency of the dehydrogenase
unit.5 In a large cohort of DBP-deficient patients enzyme activity
measurements showed that the most represented deficiency was
type III (45% of the patients), followed by type II (28%) and type
I (27%),.6 No differences in clinical signs and symptoms were
found between the three subgroups of patients. Instead, Kaplan-
Meier survival analysis found differences among the three types
of disease: type I patients had the most severe form of the disease
with a mean age of death of 6.9 months, while the mean age of

death for type II and type III patients was 10.7 months and 17.6
months, respectively.6 Our patient had a very severe form of the
disease and died when she was 8 months old. Immunoblot analy-
sis of DBP revealed the absence of the 79 and 45 kDa bands of
DBP with trace amounts of the 35 kDa component which
strongly suggests that the proband can be classified as a type 1
deficient patient.
References
1 Dieuaide-Noubhani M, Asselberghs S, Mannaerts GP, Van Veld-
hoven PP. Evidence that multifunctional protein 2, and not multi-
functional protein 1, is involved in the peroxisomal beta-oxidation of
pristanic acid. Biochem. J. 1997; 325: 367–73.
2 Jiang LL, Kurosawa T, Sato M, Suzuki Y, Hashimoto T. Physiologi-
cal role of D-3-hydroxyacyl-CoA dehydratase/D-3- hydroxyacyl-
CoA dehydrogenase bifunctional protein. J. Biochem. (Tokyo) 1997;
121: 506–13.
3 Novikov D, Dieuaide-Noubhani M, Vermeesch JR, Fournier B,
Mannaerts GP, Van Veldhoven PP. The human peroxisomal multi-
functional protein involved in bile acid synthesis: Activity measure-
Miller syndrome with novel dihydroorotate dehydrogenase
gene mutations ped_3303 587..611
Fumiko Kinoshita,1 Tatsuro Kondoh,4 Kazuhiro Komori,5 Takeshi Matsui,2 Naoki Harada,2 Akinori Yanai,1
Masafumi Fukuda,4 Kanako Morifuji3 and Tadashi Matsumoto3
1
Department of Pediatrics, Nagasaki Municipal Hospital, 2Nagasaki Laboratory, Mitsubishi Chemical Medience Corporation,
3
Department of Nursing, Nagasaki University School of Medicine, Nagasaki, 4Division of Developmental Disabilities, The
Misakaenosono Mutsumi Developmental, Medical and Welfare Center, Isahaya, 5Department of Pediatrics, Nagasaki
Hospital Agency Kamigoto Hospital, Minami-Matsuura, Japan
Key words dihydroorotate dehydrogenase (DHODH) gene, Miller syndrome, postaxial acrofacial dysostosis.
Miller syndrome (postaxial acrofacial dysostosis; OMIM
#26375) was described by Miller et al. in 1979;1 it is character-
ized by postaxial limb deficiency, cup-shaped ears, and malar
hypoplasia. The etiology of this syndrome, which is the mutation
of the dihydroorotate dehydrogenase (DHODH) gene, was estab-
lished in 2010.2 Here we report a Japanese girl with Miller syn-
drome, probably the first case in Japan, with novel compound
heterozygous mutations of the DHODH gene.
Case report
The patient, a 2-year-old Japanese girl, was the first child of
nonconsanguineous healthy parents. The pregnancy course was
Correspondence: Fumiko Kinoshita, MD, Department of Pediatrics,
Nagasaki Municipal Hospital, Shinchi 6-39, Nagasaki 850-8555,
Japan. Email: ped_kinoshita@nmh.jp
Received 20 February 2010; revised 21 September 2010; accepted 2
November 2010.
doi: 10.1111/j.1442-200X.2010.03303.x
A severe case of DBP deficiency 587
ment, deficiency in Zellweger syndrome and chromosome mapping.
Biochim. Biophys. Acta 1997; 1360: 229–40.
4 Baes M, Huyghe S, Carmeliet P et al. Inactivation of the peroxiso-
mal multifunctional protein-2 in mice impedes the degradation of
not only 2-methyl-branched fatty acids and bile acid intermediates
but also of very long chain fatty acids. J. Biol. Chem. 2000; 275:
16329–36.
5 Wanders RJA, Barth PG, Heymans HAS. Single peroxisomal
enzyme deficiencies. In: Scriver CR, Beaudet AL, Sly WS, Valle D
(eds). The Molecular and Metabolic Bases of Inherited Disease, 8th
edn. McGraw-Hill, New York, 2001; 3219–56.
6 Ferdinandusse S, Denis S, Mooyer PAW et al. Clinical and bio-
chemical spectrum of d-bifunctional protein deficiency. Ann.
Neurol. 2006; 59: 92–104.
7 Leenders F, Dolez V, Begue A et al. Structure of the gene for the
human 17beta-hydroxysteroid dehydrogenase type IV. Mamm.
Genome 1998; 9: 1036–41.
8 Barkovich AJ. Concepts of myelin and myelination in neuroradiol-
ogy. Am. J. Neuroradiol. 2000; 21: 1099–109.
9 van der Knaap MS, van der Grond J, van Rijen PC et al.
Age-dependent changes in localized proton and phosphorus
MR spectroscopy of the brain. Radiology 1990; 176: 509–
15.
uneventful. The mother and father were 20 and 25 years old,
respectively, when the girl was born. She was born at 38 weeks of
gestational age by normal vaginal delivery with weight and
length of 3480 g (+1.0 SD) and 50.0 cm (+0.6 SD), respectively.
Her weight was 10.5 kg (+1.0 SD) and height 77.2 cm (mean) at
the age of 15 months. At the age of 2 years and 5 months, her
weight was 13.0 kg (+ 0.7 SD), height 87.6 cm (mean), and head
circumference 46.7 cm (-0.7 SD). She was referred to a pediatric
department because of her limb anomalies.
She had mild micrognathia, mild malar hypoplasia, sparse
eyebrows and eyelashes, hypertelorism, down-slanting short
palpebral fissures, lower eyelid clefts, protruding and low set
small, cup-shaped ears, long philtrum, conical teeth, and anky-
loglossia (Fig. 1a). Her cleft palate was not seen. She also had
pectus excavatum and an accessory nipple near her left underarm.
Postaxial limb deficiencies included absence/hypoplasia/
dysplasia of the fifth digits in all limbs, without short forearms
(Fig. 1b–e). Bone X-rays of her hands showed the absence of
© 2011 The Authors
Pediatrics International © 2011 Japan Pediatric Society