J Inherit Metab Dis (2010 ) 33 (Suppl 3):S25–S33
DOI 10.1007/s10545-009-9012-y

CASE REPORT

Perinatal hypophosphatasia presenting as neonatal epileptic
encephalopathy with abnormal neurotransmitter
metabolism secondary to reduced co-factor
pyridoxal-5′-phosphate availability
Shanti Balasubramaniam & Frank Bowling & Kevin Carpenter & John Earl &
Jeffrey Chaitow & James Pitt & Etienne Mornet & David Sillence & Carolyn Ellaway

Received: 10 July 2009 / Revised: 21 October 2009 / Accepted: 22 October 2009 / Published online: 5 January 2010
# SSIEM and Springer 2009

Abstract We describe two neonates presenting with perinatal hypophosphatasia and severe epileptic encephalopathy resulting in death. Both had increased levels of urinary
vanillactate, indicating functional deficiency of aromatic
amino acid decarboxylase, a pyridoxal-5-phosphate (PLP)dependent enzyme required for dopamine and serotonin
biosynthesis. Clinical findings and results of subsequent

metabolic investigations were consistent with secondary
pyridoxine-deficient encephalopathy. These patients highlight the importance of tissue non-specific alkaline phosphatase in the neuronal PLP-dependent metabolism of
neurotransmitters. In addition, the disturbance of PLP
metabolism appears to underlie the predominant neurological presentation in our patients. We recommend the

Communicated by: Georg Hoffmann
S. Balasubramaniam : C. Ellaway
Genetic Metabolic Disorders Service,
The Children’s Hospital at Westmead,
Sydney, Australia
F. Bowling
Department of Biochemical Disease, Mater Health Services,
Brisbane, Australia
K. Carpenter
NSW Biochemical Genetics Service,
The Children’s Hospital at Westmead,
Sydney, Australia
J. Earl
Department of Clinical Chemistry,
The Children’s Hospital at Westmead,
Sydney, Australia
J. Chaitow
Department of Paediatric Rheumatology,
The Children’s Hospital at Westmead,
Sydney, Australia
J. Pitt
Victorian Clinical Genetics Services,
Murdoch Children’s Research Institute,
Royal Children’s Hospital Melbourne,
Melbourne, Australia

J. Pitt
Department of Paediatrics, University of Melbourne,
Melbourne, Australia
E. Mornet
Laboratoire SESEP, Centre Hospitalier de Versailles,
Le Chesnay, France
E. Mornet
Equipe EA2493,
Université de Versailles Saint-Quentin en Yvelines,
Versailles, France
D. Sillence
Department of Clinical Genetics,
The Children’s Hospital at Westmead,
Sydney, Australia
K. Carpenter : D. Sillence : C. Ellaway
Discipline of Genetic Medicine, University of Sydney,
Sydney, Australia

C. Ellaway (*)
Western Sydney Genetics Program,
The Children’s Hospital at Westmead Clinical School,
Locked Bag 4001, Westmead, NSW 2145, Australia
e-mail: Carolyne@chw.edu.au

J Inherit Metab Dis (2010 ) 33 (Suppl 3):S25–S33

S26

measurement of serum alkaline phosphatase (ALP) during
the assessment of perinatal seizures.

onstrated secondary abnormalities in intermediary metabolism consistent with pyridoxine-responsive seizures (PRSs).

Introduction

Case reports

Hypophosphatasia is a rare inherited metabolic disorder
characterised clinically by defective mineralisation of bone
and teeth and, biochemically, by deficient activity of the
tissue non-specific isoenzyme of alkaline phosphatase. The
prevalence at birth of the severe forms of the disease has
been estimated at 1:100,000 (Fraser 1957). Hypophosphatasia was initially described in a 9-week-old infant with
severe rickets and very low alkaline phosphatase (ALP)
activity in serum and tissues (Rathbun 1948). Based on the
age at diagnosis, hypophosphatasia has been classified into
six clinical subgroups: perinatal lethal, prenatal benign,
infantile, childhood and adult onset forms, and odontohypophosphatasia. Clinical variability has been reported in all
subgroups, and hypophosphatasia is best regarded as a
spectrum disorder (Fedde et al. 1996; Whyte et al. 1996).
The enzymatic deficiency results from mutations in the
liver/bone/kidney alkaline phosphatase gene (ALPL;
OMIM 171760). Laboratory diagnosis is confirmed by
reduced levels of serum alkaline phosphatase, raised levels
of urinary phospho-ethanolamine (PEA) and DNA sequencing of the ALPL gene, that detects approximately
95% of mutations in the severe (perinatal and infantile)
forms (Mornet 2007). The ALPL gene is located on
chromosome 1p36.1-34 (Greenberg et al. 1990) and
consists of 12 exons spanning 50 kb (Weiss et al. 1986).
More than 190 distinct mutations have been described,
most of which are missense mutations (79%) (http://www.
sesep.uvsq.fr/Database.html) that result in variable clinical
expression.
Patients with the perinatal lethal form present with
complications of markedly impaired mineralisation in utero
and usually succumb to respiratory distress due to hypoplastic lungs and rachitic deformities of the chest. Skincovered osteochondral spurs (Bowdler spurs) protruding
from the forearms or legs are often diagnostic of hypophosphatasia. Typical radiographic features include reduced
ossification with unusually dense, round, flattened and
butterfly shaped vertebral bodies and generalised smaller
bones. Other clinical features include apnoea, seizures and
significant shortening of the long bones (Whyte 1994). The
prenatal benign form may show spontaneous improvement
of the skeletal defects. Progressive ultrasonography has
revealed resolution of the skeletal deformities and improved
mineralisation during the third trimester of pregnancy
(Pauli et al. 1999; Wenkert et al. 2007).
In this report, we describe two neonates with perinatal
hypophosphatasia and epileptic encephalopathy who dem-

Patient 1
The patient was a full-term girl born at 39 weeks’ gestation,
with good Apgar scores after an uncomplicated pregnancy.
Her birth weight was 2.59 kg (2nd percentile) and head
circumference was 33 cm (2nd percentile). Her parents
were non-consanguineous Australians of European ancestry. She was active and fed well for her initial 4 days of life.
On day 5 she had a right-sided clonic seizure that lasted
approximately 10 min. The seizure was managed intravenously with phenobarbitone, and she required supplemental
oxygen. She was examined and treated for presumed sepsis.
The initial sepsis work up, including a lumbar puncture,
showed no evidence of infection. A cranial ultrasound was
normal.
She had a further seizure on day 7 of life. Despite
commencement of pyridoxine orally, 25 mg daily, the
seizures increased in frequency when she was 3 weeks of
age, and a loading dose of phenytoin was administered
intravenously. She was noted to have a mild metabolic
acidosis with lactic acidaemia, (lactate 10.4 mmol/l,
reference range 0.0–2.0 mmol/l). She progressed to respiratory failure necessitating intubation and ventilation and
was transferred to the neonatal intensive care unit. An
electroencephalogram (EEG) was abnormal, with a moderate burst-suppression pattern and multifocal epileptiform
discharges.
Clinical examination showed the patient to have wide
anterior and posterior fontanelles that were continuous in
the midline, but she had no other dysmorphic features. She
had generalised hypotonia and muscle weakness, with
limited antigravity movements of her limbs. Her deep
tendon reflexes were difficult to elicit. A chest X-ray
showed lytic lesions of both proximal humeri and 11 ribs
bilaterally that were short and narrow but of normal density
(Fig. 1). A modified skeletal survey, which did not include
skull radiographs, confirmed the punched-out lucencies in
the proximal humeri, and they were also present in the
femora, tibiae and the distal ulnar metaphyses (Fig. 2).
There was cupping of the long bone metaphyses. Her blood
lactate levels normalised.
Based on the patient’s clinical and radiological features
and the biochemical findings of low serum alkaline
phosphatase activity and grossly elevated levels of urinary
PEA (Table 1), a provisional diagnosis of perinatal
hypophosphatasia was made. Urinary organic acid analysis
of a urine sample obtained prior to the commencement of

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clinically asymptomatic. Their serum ALP levels were low,
and their urinary PEA levels were normal (see Table 1).
These biochemical findings were consistent with parental
heterozygosity.
Patient 2

Fig. 1 Chest X-ray of patient 1, showing lytic lesions of the proximal
humeri. The ribs numbering 11 bilaterally are short and reduced in
width but are otherwise of normal density

pyridoxine administration and while the patient was not on
catecholamine support revealed the presence of vanillactate,
compatible with secondary pyridoxine deficiency and
consequent aromatic amino acid decarboxylase deficiency.
Levels of other dopa-metabolites [homovanillic acid
(HVA), vanillylmandelic acid (VMA), 3,4-dihyroxyphenylacetate] were normal, therefore excluding common secondary causes of increased vanillactate excretion such as L-dopa
or catecholamine treatment and neonatal tyrosinaemia. There
was normal excretion of 2-amino-adipic semi-aldehyde and
pipecolic acid, which excluded amino-adipic semi-aldehyde
dehydrogenase deficiency secondary to mutations in the
PNPO gene as a cause of seizures. An EEG obtained
2 weeks after commencement of pyridoxine therapy showed
some improvement, with no burst suppressions, but it
remained abnormal, with an excess of sharp waves for her
age. Her condition remained clinically unstable, with
frequent desaturations and wide fluctuations in her blood
pressure, ranging from mean arterial pressures of 110 mmHg
to 30 mmHg. Her neurological status deteriorated. She
became more obtunded 6 days after ventilation, with
ongoing episodes of desaturation, tachycardia and hypertension, but no overt seizures. The pyridoxine dose was
increased to 100 mg daily orally, and then to 100 mg
intravenously. She succumbed to her illness at 5-weeks
of life.
Cerebrospinal fluid (CSF) analysis, performed in the
peri-mortem period, revealed elevated levels of PEA, while
levels of other amino acids were normal and that of 5hydroxyindole-acetic acid (5-HIAA) was mildly reduced
(Table 2). Biopterin was borderline low, and there were
additional peaks from pyridoxine metabolites, due to the
pharmacological doses administered. Both parents were

This patient was a boy born to healthy non-consanguineous
parents. At birth he was found to have a malformed rib cage
(Fig. 3), multiple skeletal (Fig. 4) anomalies and rhizomelic
shortening of the limbs. The radiographs showed bowing of
some long bones and slender and demineralised ribs. The
skeletal survey was consistent with changes seen in
infantile rather than perinatal lethal hypophosphatasia.
Ineffective respiratory effort compounded by refractory
seizures from day 1 of life necessitated mechanical
ventilation. Seizure control was initially not achieved with
phenobarbitone and clonazepam. The EEG showed a burstsuppression pattern. Pyridoxine was administered intravenously at 50 mg daily, which led to the cessation of seizure
activities. A cranial ultrasound was normal. A maintenance
dose of pyridoxine, (10 mg/day) was continued, while all
other anticonvulsants were ceased. An EEG performed
several days after the commencement of pyridoxine
treatment showed the disappearance of the burstsuppression pattern. The ALP level was low (<5 IU/l,
reference range 100–400 IU/l) (Table 1). Urinary levels of
PEA and vanillactate were elevated, CSF 5-HIAA and HVA
were reduced, and 3-O-methyldopa level was elevated
(Table 2). The baby died on day 7 of life. The mother had
poor dentition, with badly decayed teeth. She had no

Fig. 2 a, b Radiographs of both lower limbs of patient 2, showing
punched-out metaphyseal lucencies in the proximal and distal femora
and tibiae

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Table 1 Serum levels of ALP activity and urinary phospho-ethanolamine in the patients and their parents. Abbreviation: NA, not available
Family members

Age

ALP
(IU/l)

Reference ranges
(IU/l)

Urinary phospho-ethanolamine

Reference ranges
(µmol/mmol creatinine)

Patient 1
Father
Mother
Patient 2
Mother
Father

3 weeks
38 years
30 years
1 week
39 years
NA

14
46
53
<5
16
NA

160–400
80–355
80–355
100–400
35–110
NA

1,300
9
17
1015
39
NA

10–20
5–18
5–18
0–70
5–20
NA

history of fractures. She had low levels of serum ALP and
mildly elevated urinary PEA (Table 1). We were unable to
obtain blood or urine samples from the father.

CSF neurotransmitter analysis
Cerebrospinal fluid analysis for 5-HIAA, HVA and 3-Omethyldopa were measured by high performance liquid
chromatography (HPLC) with electrochemical detection.

Methods
ALPL gene mutation detection by DNA sequencing
analysis

Urine phospho-ethanolamine quantitation
Urinary PEA was quantitated by amino acid analysis using
ion exchange chromatography and post-column ninhydrin
detection.
Urine vanillactate detection
Urinary vanillactate was detected by standard organic acid
analysis using gas chromatography–mass spectrometry
following ethyl acetate extraction and formation of trimethylsilyl derivatives.
Urinary 2-amino-adipic semi-aldehyde and pipecolic acid
detection
Urinary 2-amino-adipic semi-aldehyde and pipecolic acid
were measured by gas chromatography–mass spectrometry
after derivatisation with ethoxyamine and propylchloroformate (Pitt and Eggington 2007).
Table 2 Cerebrospinal fluid
levels of specific catecholamines
and amino acids. Abbreviation:
NA, not available

Primer sequences were designed from the intronic sequences obtained from Genbank (accession numbers D87874 to
D87888), Orimo et al. 1997, and from Mornet et al. 1998).
These primers allowed analysis of the entire coding region
of the ALPL gene. Polymerase chain reaction (PCR) was
performed in a final volume of 50 µl with 30 pmol of each
primer, 250 µM of each deoxyadenosine triphosphate
(dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP) and deoxythymidine triphosphate
(dTTP), 1 unit of gold Taq DNA polymerase (Perkin-Elmer,
Foster City, CA, USA] and 1–3 mM magnesium chloride
(MgCl2), depending on the exon amplified. The mixtures
were heated at 95°C for 10 min and subjected to 30 cycles
of 1 min at 95°C, 1 min at the annealing temperatures and
40 s at 72°C. The PCR products were purified and
sequenced by the direct sequencing method using the ABI
Prism Dye Terminator cycle sequencing ready reaction kit
with AmpliTaq DNA polymerase, FS (Perkin-Elmer) and

Metabolites

Patient 1

Reference ranges

Patient 2

Reference ranges

5-HIAA
HVA
3-O-methyldopa
Threonine
Serine
Glycine
Phospho-ethanolamine
Total biopterin
Tetrahydrobiopterin
Neopterin

0.26 µmol/l
0.69 µmol/l
NA
38 µmol/l
33 µmol/l
5 µmol/l
109 µmol/l
22 nmol/l
NA
NA

>0.38 µmol/l
>0.54 µmol/l
NA
21–121 µmol/l
32–82 µmol/l
4–14 µmol/l
3–8 µmol/l
25–45 nmol/l
NA
NA

0.01 µmol/l
0.05 µmol/l
1.75 µmol/l
67 µmol/l
32 µmol/l
9 µmol/l
320 µmol/l
NA
21 nmol/l
25 nmol/l

0.12–0.5 µmol/l
0.3–1.1 µmol/l
<0.3 µmol/l
21–121 µmol/l
32–82 µmol/l
5–8 µmol/l
NA
NA
10–30 nmol/l
7–65 nmol/l

J Inherit Metab Dis (2010 ) 33 (Suppl 3):S25–S33

Fig. 3 Chest X-ray of patient 2. The ribs are slender, reduced in width
and flared anteriorly

migrated on to an ABI Prism 310 electrophoresis system
(Applied Biosystems, USA).

Results
DNA from patient 1 was not available, but her parents were
both heterozygotes for mutations of the ALPL gene. The father
was heterozygous for a frameshift mutation that resulted in a
premature stop codon, c.891C>A (p.Y297X). This mutation
has not been previously described in hypophosphatasia. The
mother was heterozygous for the mutation, c.1471G>A
(p.G491R), which has been previously reported in the severe
perinatal lethal form. The combination of a truncating
mutation and a severe mutation in the parents was consistent
with the inheritance of both these alleles in patient 1 and the
diagnosis of perinatal lethal hypophosphatasia. The second
patient was found to have a pathogenic mutation, c.318G>C
(p.Q106H) that involves the homodimer interface, a critical
domain. In addition, the mutation may create an exon splicing
enhancer (ESE), which may result in alternative splicing. The
results were compatible with homozygosity for the p.Q106H
mutation or double heterozygosity, which could occur in
association with a deletion in the second allele. The patient’s
parents needed to be tested so that we could distinguish
homozygosity from hemizygosity. Unfortunately, the father
was not available for testing.

Discussion
Hypophosphatasia is a rare inherited skeletal dysplasia with
highly variable clinical expression. It is caused by muta-

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tions in the liver/bone/kidney alkaline phosphatase gene
(ALPL; OMIM 171760), which encodes tissue non-specific
alkaline phosphatase (TNAP).
Clinical expression of hypophosphatasia ranges from
stillbirth with demineralised bone to pathological fractures
in adult life (Whyte 1994; Mornet 2007). The autosomal
recessive perinatal lethal form is the most severe and is
characterised by impaired bone mineralisation in utero,
shortening of the long bones, respiratory distress, apnoea
and seizures (Mornet 2007). Pauli and colleagues reported
in 1999 a sixth form of hypophosphatasia; a prenatal benign
form that demonstrates spontaneous postnatal improvement. This “bent but not broken” phenotype (Pauli et al.
1999) has a postnatal course that is much less severe than
either the perinatal lethal or infantile forms. The inheritance
of the prenatal benign phenotype is consistent with
autosomal dominant inheritance due to the dominantnegative effect of ALPL gene mutations on TNAP catalytic
activity (Lia Baldini et al. 2001; Brun-Heath et al. 2008).
The skeletons of both patients in this study were assessed as
being less severely affected than in typical perinatal lethal
cases, patient 2 even more so than patient 1. Therefore, the
impact of the neonatal encephalopathy was surprising.
At least four genes encode the isoenzymes of ALP in
humans (Harris 1990). Three of these genes, clustered on
chromosome 2q34-47, direct synthesis of the tissue-specific
ALP isoenzymes of the intestine, placenta and germ cells.
The fourth gene, ALPL, located at chromosome 1p36.1-34,
has expression that is more widespread than originally
recognised, including the developing brain (Fonta et al.
2005). The enzyme cleaves extracellular substrates such as
pyridoxal-5-phosphate (PLP), PEA, and inorganic pyrophosphates (Whyte 1994). TNAP is normally abundant in
osteoblasts and chondrocytes, where it is coupled to the cell
surface and functions as an ectoenzyme. In bone, inorganic
Fig. 4 Left forearm of patient 2,
showing bowing of the
proximal ulna

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pyrophosphate retards the growth of nascent hydroxyapatite
crystals present in bone matrix vesicles, which are the
initial sites of mineral deposition (Anderson et al. 2004;
Cole 2008). Intracellular accumulation of inorganic pyrophosphates accounts for impaired skeletal mineralisation
(Whyte et al. 1996). Human TNAP also plays an important
role in vitamin B6 metabolism, by dephosphorylating PLP
to pyridoxal (PL) and is necessary for cellular uptake. This
is indicated by the increased plasma concentration of PLP
(Iqbal et al. 1998; Litmanovitz et al. 2002) and reduced
plasma concentration of pyridoxal in severe forms of
hypophosphatasia (Whyte et al. 1988).
Vitamin B6 is a water-soluble vitamin that is present in
the body as six vitamers: pyridoxine, pyridoxamine,
pyridoxal and their 5′-phosphorylated esters, with pyridoxal
being the only vitamer with co-factor activity (Fig. 5).
Animal-derived vitamin B6 consists primarily of phosphorylated pyridoxal and pyridoxamine, whilst the plant-derived
forms consist largely of free and bound pyridoxine (Surtees
et al. 2006). Intestinal alkaline phosphatases convert
phosphorylated B6 vitamers to their bases, which are
subsequently absorbed from the upper small intestine by a
carrier-mediated system into the portal circulation and liver
(Said 2004; Surtees et al. 2006). In the liver, pyridoxal
kinase phosphorylates pyridoxine, pyridoxamine and pyri-

doxal to their 5′-phosphate esters, whilst pyridoxamine-5′phosphate oxidase (PNPO) oxidises pyridoxine phosphate
and pyridoxamine phosphate to form PLP. Albumin-bound
PLP is released into the circulation and forms approximately 60% of circulating vitamin B6. Human TNAP dephosphorylates circulating PLP at the choroid plexus, releasing
pyridoxal for transportation into the cerebrospinal fluid by
an active transport mechanism (Surtees et al. 2006). PL is
the form of vitamin B6 that crosses cell plasma membranes
to be rephosphorylated to PLP in the neurons. Pyridoxine
kinase catalyses the phosphorylation of pyridoxal, pyridoxine and pyridoxamine, whilst pyridoxine and pyridoxamine
phosphate are then oxidised by PNPO to form PLP (Kang
et al. 2004; Surtees et al. 2006). PLP is the active
metabolite of vitamin B6 and is an essential coenzyme for
synthesis of various neurotransmitters and biogenic amines
(Whyte et al. 1988). It acts as a co-factor for more than 100
apoenzymes. The corresponding holoenzymes catalyse
diverse reactions such as transamination, decarboxylation,
racemisation, elimination and replacement. In the brain,
PLP-dependant enzymes [aromatic amino acid decarboxylase, branched-chain amino acid 2-oxoglutarate aminotransferase, gamma-aminobutyric acid (GABA) transaminase,
glutamate decarboxylase, glycine cleavage enzyme, kynureninase, kynurenine aminotransferase and L-serine race-

Tryptophan

Tyrosine

5-HTP

L-DOPA

3-O-MD

DA

NA

EPI

MHPG

VMA

Vanillactate

AADC
TNAP

Serotonin
PL

PLP

5-HIAA
HVA

Note: Arrows in red indicate biochemical findings in AADC deficiency
5-HTP=hydroxytryptophan; 3-O-MD= 3 - O- methyldopa; AADC= aromatic L- amino acid decarboxylase; DA=dopamine; LDOPA= L-dopamine; NA=noradrenaline; EPI=epinephrine; 5-HIAA= 5-hydroxyindole acetic acid; HVA= homovanillic acid;
MHPG= 3-methoxy-4 hydroxyphenylglycol; VMA= vanillylmandelic acid; PL= pyridoxine; PLP= prridoxal-5-phosphate;
TNAP=Tissue Non Specific Alkaline Phosphatase.

Fig. 5 Monoamine metabolism pathway and secondary aromatic amino acid decarboxylase (AADC) deficiency

J Inherit Metab Dis (2010 ) 33 (Suppl 3):S25–S33

mase] are involved in the metabolism of dopamine,
serotonin, glutamate, glycine, GABA, D-serine and taurine
(Surtees et al. 2006). Hence, inadequate levels of PLP in the
neurons may lead to neurological dysfunction, particularly
epilepsy.
Neonatal seizures have previously been described in
hypophosphatasia and were believed to be related to cranial
deformity, haemorrhage or hypoxia (Whyte et al. 1988).
These complications were excluded in both our patients.
Takahashi and colleagues suggested in 1984 that high
endogenous concentrations of PEA, a natural substrate of
TNAP, may also be epileptogenic. The association of
pyridoxine-responsive seizures (PRSs) with perinatal hypophosphatasia was first described in 1967 (Bethenod et al.
1967), with seven similar cases of PRSs with hypophosphatasia (HPP) subsequently reported (Whyte et al. 1988;
Bethenod et al. 1967; Litmanovitz et al. 2002). Reports of
at least ten additional hypophosphatasia patients with
neonatal seizures have been published, mostly as abstracts,
where pyridoxine or PLP were not administered. Whether
seizures in these patients were pyridoxine- or PLPresponsive/dependent is unclear, as the anticonvulsive
action of vitamin B6 was not known at that time or it was
not given (Baumgartner-Sigl et al. 2009). The pathophysiology of PRSs in hypophosphatasia appears to be reduced
brain PLP concentrations, decreasing the seizure threshold.
Baumgartner-Sigl et al. 2009 described a 7-month-old girl
with infantile hypophosphatasia, who uniquely presented as
a neonate with PRSs but did not have skeletal abnormalities. One other report briefly describes infantile hypophosphatasia presenting with PRSs, which was lethal when the
patient was aged 3 months but had relatively mild bone
disease (Posen et al. 1997). Like our patient, all reported
HPP patients with neonatal seizures died within 18 months.
Urinary levels of vanillactate were elevated in both our
patients. This metabolite indicates functional deficiency of
aromatic amino acid decarboxylase (AADC), a PLPdependent enzyme involved in dopamine and serotonin
metabolism (Fig. 5). Patient 1 was therefore examined for
amino-adipic semi-aldehyde dehydrogenase deficiency,
which causes secondary pyridoxine deficiency. The results
of these investigations were essentially normal, as
evidenced by normal excretions of 2-amino-adipic semialdehyde. Levels of urinary vanillactate are also increased
in primary AADC deficiency, due to mutations in the gene
encoding this enzyme. Analysis of the CSF of both patients
showed reduced 5-HIAA levels. Reduced HVA and
elevated 3- methoxytyrosine (3-O-methyl-dopa), and mildly
raised levels of glycine, were observed in the second
patient. These abnormal metabolites may have been
indicators of deficient flux through multiple pathways
involving PLP-dependent enzymes, including AADC
(Clayton 2006). If AADC activity is measured in plasma,

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it may be normal, as it is a secondary dysfunction resulting
from co-factor inadequacies, rather than a true enzymatic
deficiency, in which case the HVA and 5-HIAA are more
markedly reduced. Mutation analysis of the AADC gene
can be performed to confirm the diagnosis of primary
AADC and distinguish it from secondary dysfunction.
Recessively inherited deficiency of AADC (OMIM
608643) results in a severe neurometabolic disorder with
developmental delay, movement disorder with truncal
hypotonia, limb hypertonia, dystonia, athetosis and hypokinesia, oculogyric crises and vegetative symptoms such as
hypersalivation, nasal congestion, excessive sweating and
gastro-oesophageal reflux disease. Patients display a typical
pattern of neurotransmitters in CSF, with a marked
reduction of the stable degradation products of dopamine
and serotonin pathways, HVA and 5-HIAA, as well as
elevation of the precursors of dopamine and serotonin,
laevodopa, 5-hydroxytryptophan and 3-O-methyldopa (3OMD) resulting from methylation of accumulating laevodopa (Manegold et al. 2009). A similar pattern was recently
described in patients with mutations in the PNPO gene
(Mills et al. 2005). These patients have a secondary AADC
deficiency due to a defect in the synthesis of pyridoxal
phosphate. In contrast to our patients, there are only few
reported AADC patient with epileptic seizures (Ito et al.
2008; Swoboda et al. 2003; Manegold et al. 2009). More
often, the paroxysmal movement disorder in AADC
deficiency is misinterpreted as epileptic seizures. Plasma
and CSF concentrations of PLP and pyridoxal were not
measured simultaneously in either patient, but these would
be expected to show increased plasma levels of PLP and
reduced or undetectable amounts of pyridoxal. Both our
patients showed disappearance of baseline burst suppression after commencement of pyridoxine therapy. The
improvements in the EEG, however, was more evident in
the second patient, and this could perhaps be attributed to
the higher dose of intravenously administered pyridoxine
that was used, in contrast to a smaller dose of pyridoxine
given orally to the first patient. We also speculated that the
Q106H mutation is possibly milder than the G491R
mutation.
Carriers of the severe forms of hypophosphatasia are
usually clinically unaffected. They may, however, show
modestly reduced levels of serum ALP, as did the parents of
our patients, and increased urinary PEA levels, observed in
the mother of the second patient (Mornet 2007). Important
in our understanding of hypophosphatasia is the temporal
variability of the bone disease (Whyte et al. 2006; Cole
2008). Progressive improvements in limb shortening, long
bone bowing and mineralisation may occur during the third
trimester of pregnancy (Pauli et al. 1999; Wenkert et al.
2007). Recognition of this possibility is crucial and
emphasises the need for expert assessment and careful

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counselling on the natural history of hypophosphatasia in
each case of perinatal diagnosis (Cole 2008). The progressive improvements of affected patients from infancy to
adulthood may be attributable to epigenetic factors involved
in the variable expression of the disease or the temporal
requirements for ALP during development (Mornet 2007).
An alternative explanation for the spontaneous remission of
skeletal deformities in the prenatal benign form is that
maternal ALP compensates, to a degree, via foetal–
maternal exchanges, but this is primarily observed when
the mother is heterozygous for a mutation with a dominant
negative effect (Pauli et al. 1999; Moore et al. 1999;
Mornet 2007). It has been reported that maternal serum
ALP activity increases during approximately the last
trimester of pregnancy, when the placental ALP isoenzyme
appears in the maternal circulation. This increase could
mask the hypophosphatasaemia in the mother (Whyte
1995). On the other hand, if the mutation is inherited from
the father, normal maternal ALP levels will prevent
manifestation of the disorder in the foetus.
Currently, there is no specific treatment for hypophosphatasia. Management is directed towards preventing or
correcting the symptoms and complications. Good
responses to treatment with pyridoxine and pyridoxal
phosphate have been described for the control of neonatal
convulsions with burst suppressions on EEG or infantile
spasms with hypsarrhythmia (Clayton 2006). The wide
clinical heterogeneity and the rarity of the disease render
controlled clinical trials almost impossible. Preliminary
results have suggested that dietary phosphate restriction
could be beneficial, because patients are hyperphosphataemic and inorganic phosphate is a natural inhibitor of TNAP
(Cole 2008; Wenkert et al. 2007). Treatments with zinc and
magnesium, which act as catalytic ions of the enzyme,
showed inconsistent results.
Bone marrow transplantation has been performed in the
more severe infantile cases, in a small number of patients.
The results suggested that the effects may be transient, with
bone lesions possibly recurring 6 months after transplantation (Whyte et al. 2003). Donor bone fragments and
marrow may provide precursor cells for distribution and
engraftment in the skeletal microenvironment that form
TNAP-replete osteoblasts and may improve mineralisation
(Whyte et al. 2003). Further trials with improved transplant
protocols may enable the establishment of this treatment
modality (Cole 2008).
Finally, enzyme replacement therapy appears to be
the most promising potential therapy in the near future.
The first successful treatment of TNAP knockout mice,
using exogenous administration of recombinant ALP
that targeted mineralised tissues, has been reported
(Millan et al. 2007). Human studies will clarify the
efficacy of enzyme replacement therapy in substituting

endogenously deficient TNAP and potentially ameliorating this disorder.

Conclusion
We have described two patients with autosomal recessive
perinatal hypophosphatasia with epileptic encephalopathy
and central hypoventilation, and autonomic instability in
one of them. Of interest, both patients had relatively mild
defects in skeletal mineralisation, in comparison with the
severe extraskeletal features. In both patients progressive
encephalopathy, refractory to therapy, resulted in perinatal
death.
Deactivating mutations in the ALPL gene that encodes
TNAP may lead to secondary AADC deficiency, due to
reduced co-factor pyridoxal-5-phosphate availability. Both
patients had raised levels of urinary vanillactate and
reduced levels of CSF 5-HIAA, whilst reduced HVA and
raised 3-O-methyldopa levels were also documented in the
second case. In addition, both patients exhibited burst
suppressions on EEG that responded to pyridoxine
therapy.
We recommend that assessment of any neonate suffering
PRSs should include the measurement of serum ALP.
Further detailed metabolic studies comprising measurements of plasma and CSF PL and PLP levels in cases of
hypophosphatasia should be conducted. Accurate clinical
diagnosis is crucial. Neurological complications should be
anticipated and, if present, we suggest the early institution
of treatment with pyridoxine or pyridoxal phosphate, as it
may prevent or ameliorate seizures. Furthermore, we
emphasise the importance of a treatment trial of pyridoxine
and/or pyridoxal phosphate in neonates with intractable
seizures. Further studies involving complementary
approaches corroborating clinical data evaluation, sitedirected mutagenesis and computer–assisted threedimensional modelling of the ALPL gene may provide a
more profound elucidation of genotype–phenotype correlation. Greater emphasis should be placed on detailed
molecular characterisation to guide clinical management,
genetic counselling of the family and prenatal testing for
future pregnancies.

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