Download Cerebellar ataxia with elevated cerebrospinal free sialic acid

doi:10.1093/brain/awn355
Brain 2009: 132; 801–809
| 801
BRAIN
A JOURNAL OF NEUROLOGY
Cerebellar ataxia with elevated cerebrospinal
free sialic acid (CAFSA)
F. Mochel,1,2 F. Sedel,3, A. Vanderver,4, U. F. H. Engelke,5 J. Barritault,6 B. Z. Yang,2
B. Kulkarni,4 D. R. Adams,7 F. Clot,1 J. H. Ding,2 C. R. Kaneski,2 F. W. Verheijen,8
B. W. Smits,9 F. Seguin,6 A. Brice,1,10 M. T. Vanier,11 M. Huizing,7 R. Schiffmann,2
A. Durr1,10 and R. A. Wevers5
1
2
3
4
5
6
7
8
9
10
11
INSERM UMR S679, Hôpital de la Salpêtrière, Paris, France
Institute of Metabolic Disease, Baylor Research Institute, Dallas, USA
Fédération des Maladies du Système Nerveux and Reference Center for Lysosomal diseases, Hôpital de la Salpêtrière, Paris, France
Children’s National Medical Center, Children’s Research Institute, Center for Genetic Medicine, Washington, DC, USA
Radboud University Nijmegen Medical Center, Laboratory of Pediatrics and Neurology, Nijmegen, The Netherlands,
INSERM U927, Université de Poitiers, Hôpital La Milêtrie, Poitiers, France
Medical Genetics Branch, National Human Genome Research Institute, NIH, Bethesda, MD, USA
Department of Clinical Genetics, Erasmus Medical Center, Rotterdam, The Netherlands
Radboud University Nijmegen Medical Center, Department of Neurology, Nijmegen, The Netherlands
Département de Génétique et Cytogénétique, Hôpital de la Salpêtrière, Paris, France
INSERM U820, Faculté de Médecine Lyon-RTH Laennec, Lyon, France
*These authors contributed equally to this study.
Correspondence to: Dr Fanny Mochel,
INSERM UMR S679, Hôpital La Salpêtrière,
47 Bld de l’Hôpital,
Bâtiment Nouvelle Pharmacie - 4ème étage,
75013 Paris, France
E-mail: [email protected]
In order to identify new metabolic abnormalities in patients with complex neurodegenerative disorders of unknown aetiology,
we performed high resolution in vitro proton nuclear magnetic resonance spectroscopy on patient cerebrospinal fluid (CSF)
samples. We identified five adult patients, including two sisters, with significantly elevated free sialic acid in the CSF compared
to both the cohort of patients with diseases of unknown aetiology (n = 144; P _ 0.001) and a control group of patients with
well-defined diseases (n = 91; P50.001). All five patients displayed cerebellar ataxia, with peripheral neuropathy and cognitive
decline or noteworthy behavioural changes. Cerebral MRI showed mild to moderate cerebellar atrophy (5/5) as well as white
matter abnormalities in the cerebellum including the peridentate region (4/5), and at the periventricular level (3/5). Twodimensional gel analyses revealed significant hyposialylation of transferrin in CSF of all patients compared to age-matched
controls (P50.001)—a finding not present in the CSF of patients with Salla disease, the most common free sialic acid storage
disorder. Free sialic acid content was normal in patients’ urine and cultured fibroblasts as were plasma glycosylation patterns
of transferrin. Analysis of the ganglioside profile in peripheral nerve biopsies of two out of five patients was also normal.
Sequencing of four candidate genes in the free sialic acid biosynthetic pathway did not reveal any mutation. We therefore
identified a new free sialic acid syndrome in which cerebellar ataxia is the leading symptom. The term CAFSA is suggested
(cerebellar ataxia with free sialic acid).
Received November 10, 2008. Revised November 28, 2008. Accepted December 2, 2008. Advance Access publication January 19, 2009
ß The Author (2009). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.
For Permissions, please email: [email protected]
802
| Brain 2009: 132; 801–809
F. Mochel et al.
Keywords: cerebellar ataxia; free sialic acid; cerebrospinal fluid; neurometabolic disorder; nuclear magnetic resonance spectroscopy
Abbreviations: CAFSA = cerebellar ataxia with free sialic acid; CMAS = CMP-Neu5Ac synthase; CSF = cerebrospinal fluid;
HIBM = hereditary inclusion body myopathy; Neu5Ac = N-acetylneuraminic acid; NMRS = nuclear magnetic resonance spectroscopy;
NPL = Neu5Ac pyruvate lyase; SASD = free sialic acid storage diseases
Introduction
In vitro Nuclear Magnetic Resonance spectroscopy (NMRS) is a validated biochemical tool for metabolic analyses of human body fluids
and diagnosis of inborn errors of metabolism in children and adults.
The technique is of special interest because it requires minimal
sample preparation, it can simultaneously detect compounds of different nature and it offers structural information on the metabolites
present in body fluids. In the last decade, in vitro NMRS contributed
to the identification of new inborn errors of metabolism, some of
which are amenable to therapeutic intervention (Moolenaar
et al., 2003; Engelke et al., 2004, 2008; Oostendorp et al., 2006).
A number of neurological metabolic disorders are defined by
elevation of key metabolites in the cerebrospinal fluid (CSF). We
hypothesized that NMRS could allow the identification of small
metabolites in the CSF of patients with complex neurodegenerative
disorders for which extensive metabolic and genetic work-up was
negative. As a result, we identified a new metabolic entity named
CAFSA (cerebellar ataxia with free sialic acid), which extends the
range of human diseases involving free sialic acid metabolism.
Materials and Methods
Children and adults from three referral centres for neurogenetics and
neurometabolism were enrolled in clinical protocols approved by the
local ethics committees of the Assistance Publique des Hôpitaux de
Paris, France, the National Institutes of Neurological Disorders and
Stroke, Bethesda, MD, USA and the Radboud University Nijmegen
Medical Center, the Netherlands. Written informed consent was
obtained for all patients or their legal guardians.
Patients’ cohorts
Two hundred thirty five patients with progressive and complex neurological diseases were included in the study, with ages at examination
ranging from 1 to 80 years. Patients with disorders of unknown aetiology (n = 144) were classified according to the leading neurological
symptom: psychomotor retardation (n = 17), cerebellar ataxia
(n = 25), spastic paraplegia (n = 9), parkinsonism or other extrapyramidal manifestations (n = 31), neuropathy (n = 7), psychiatric symptoms
(n = 13) and leukodystrophy (n = 42). The disease control group consisted of 91 patients with similar clinical presentation but well-defined
clinical diagnoses that can be classified as (i) hypomyelinating diseases:
Salla disease, Pelizaeus-Merzbacher disease, CACH/Vanishing White
Matter disease, hypomyelination hypogonadotropic hypogonadism
and hypodontia syndrome (4H syndrome); (ii) demyelinating diseases:
Alexander disease, Krabbe disease, L-2-hydroxy glutaric aciduria, adult
polyglucosan body disease, leukoencephalopathy with brain stem and
spinal cord involvement and lactate elevation (LBSL), X-linked adrenoleukodystrophy, megalencephalic leukoencephalopathy with subcortical cysts, adult autosomal dominant leukodystrophy with Lamin B1
duplication, cerebrotendinous xanthomatosis; (iii) genetic diseases
that can affect the white matter: Wilson disease, respiratory chain
defects (mutations in nuclear and mitochondrial genes), Fabry disease,
tetrahydrobiopterin deficiency, type 3 Gaucher (chronic neuronopathic)
disease, Niemann-Pick type C disease, spastic paraplegia 11 and 15,
chromosomal abnormalities (Turner syndrome, 18q ter deletion);
(iv) genetic diseases that affect the basal ganglia: neuroacanthocytosis,
Fahr disease, Aicardi-Goutières syndrome; (v) genetic diseases that
affect the cerebellum: spinocerebellar ataxia 17, ataxia with oculomotor apraxia type 1; (vi) acquired diseases affecting the white matter:
multiple sclerosis, corticobasal degeneration and antiphospholipid
syndrome; (vii) patients with progressive conditions such as Alzheimer
disease, frontotemporal dementia, neurosarcoidosis and (viii) patients
with non-neurodegenerative neurological condition such as normal pressure hydrocephalus, stroke, Korsakoff syndrome, fish odour syndrome.
A wide panel of metabolic and genetic investigations was performed
in the cohort of 144 patients and showed no abnormality
(Supplementary methods).
Proton NMRS of body fluids
In order to identify new metabolic abnormalities, CSF was stored at
80 C waiting for serial proton NMRS analyses. In case of abnormal
findings in CSF, urine and plasma samples were also obtained from
patients and stored at 80 C. CSF, urine and plasma samples were
prepared for NMRS with minimal handling (Supplementary methods)
(Engelke et al., 2004; Mochel et al., 2007).
Investigation of sialic acid metabolism
Following significant findings by NMRS in the CSF of five patients,
further investigations were conducted. In addition to NMRS, urinary
free and bound sialic acid levels were determined by a quantitative
colorimetric assay as previously described (Romppanen and Mononen,
1995). Free and total sialic acid levels were also measured in cultured
skin fibroblasts (Kleta et al., 2003).
All exons and their surrounding intron/exon boundaries of four
candidate genes of the sialic acid biosynthetic pathway were PCR
amplified from genomic DNA and analysed by bi-directional direct
sequencing: the SLC17A5 gene (GenBank NM_012434), Neu5Ac pyruvate lyase (NPL, GenBank NM_030769), CMP-Neu5Ac synthase (CMAS,
GenBank NM_018686), as well as exon 5 of GNE (GenBank NM_
005476), coding for the allosteric site of UPD-GlcNAc 2-epimerase.
Total RNA was also isolated from confluent fibroblast cultures with
and without cycloheximide treatment (Supplementary methods) and
converted into two overlapping SLC17A5 cDNA fragments for subsequent bi-directional sequencing (Supplementary methods).
Proteomics studies of CSF
We performed two-dimensional gel electrophoresis, as well as MS and
MS/MS for protein and glycoform identification in patients’ CSF
(Supplementary methods). The volume and intensity of spots of interest
were determined and automated calculation of a ratio of asialotransferrin
to total transferrin was obtained as previously described (Vanderver
et al., 2005, 2008). 2-DG analysis and ratio calculation were performed
by an investigator (B.K) blinded to the values of free sialic acid in CSF.
Ataxia and free sialic acid
Gangliosides analyses
Due to the presence of a peripheral neuropathy, two out of five
patients have had a sural nerve biopsy, which was studied by routine
light and electron microscopy. Analysis of the ganglioside patterns
were performed on peripheral nerve tissue stored at 80 C as
described in a previous study (Timmons et al., 2006).
Statistical analysis
To compare the ratio of asialotransferrin to total transferrin between
the different patient groups, an analysis of covariance (ANCOVA) was
performed with age as a covariate. The Bonferroni method was used
to adjust the P-values associated with the multiple comparisons
between the age-adjusted means.
Results
Isolated elevation of free sialic acid
in the CSF of five patients
Among the cohort of 144 patients with complex neurological
disorders of unknown aetiology, high resolution in vitro
Brain 2009: 132; 801–809
| 803
proton NMRS of CSF revealed an increased concentration
of free sialic acid in five patients, from four families. The onedimensional proton NMR spectrum of free sialic acid, also called
N-acetylneuraminic acid (Neu5Ac), is characterized by the presence of a main peak at 2.05 ppm, corresponding to the methyl
group of free sialic acid, associated with smaller peaks around
1.85 and 2.26 ppm corresponding to the pyranose ring protons
of the carbon-3 atom (Fig. 1A). The two-dimensional proton
NMR spectrum confirms that these smaller peaks are coupled
and therefore belong to the same molecule of free sialic
acid (Fig. 1A).
The mean value of free sialic acid in the CSF of the CAFSA
patients was 43.4 11.0 mmol/l, ranging from 35.6 to 67 mmol/l
(Fig. 1B), and was highly significantly increased compared to the
cohort of patients with diseases of unknown aetiology
(8.2 4.2 mmol/l, P50.001) as well as to the control cohort of
patients with well-defined diseases (9.9 5.6 mmol/l, P50.001)
(Fig. 1B). The mean values in the two cohorts are similar to
those found in a previous study where free sialic acid was measured by high performance liquid chromatography in a small group
of normal controls (Hayakawa et al., 1993). As expected, patients
with Salla disease (n = 3), a well-known disease involving free
Figure 1 Identification of elevated free sialic acid by NMR spectroscopy (NMRS) in the CSF of five patients. (A) One-dimensional 1H
(upper) and two-dimensional 1H–1H COSY (lower) 500 MHz spectrum of the CSF of CAFSA Patient 2. The cross peaks of the H3eq
(equatorial) and H3ax (axial) protons in Neu5Ac are connected by dashed lines. The structure represents the beta-anomer of
N-acetylneuraminic acid (=free sialic acid or Neu5Ac). (B) Values of free Neu5Ac in the CSF of the 235 patients cohort, including
CAFSA and Salla patients. The elevation of free sialic acid is even greater in the CAFSA patients than in the Salla patients. Note that
higher free Neu5Ac levels can be observed in the first 4–6 months of life (data not shown).
804
| Brain 2009: 132; 801–809
sialic acid [OMIM 604369; (Verheijen et al., 1999)], had elevation
of free sialic acid in CSF as well although to a lesser degree
(31.3 4.9 mmol/l) (Fig. 1B).
Brain tumours and pyogenic meningitis, two reported
conditions leading to elevation of free sialic acid in CSF, were
ruled out. In the case of our five patients, the elevation of free
sialic acid was restricted to their CSF. Free sialic acid was indeed
normal in urine and plasma, unlike Salla patients who usually
have a marked elevation of free sialic acid in their urine. Free
and total sialic acid was also normal in patients’ cultured skin
fibroblasts. Sequencing of all exons, as well as exon–intron junctions, of SLC17A5, mutated in patients with free Sialic Acid
Storage Diseases (SASD) (Verheijen et al., 1999), did not reveal
any mutation. RT-PCR did not display any abnormal splicing
variants, even when cycloheximide was added to the cultured
media in order to inhibit non-sense mediated decay. Sequencing of exon 5 of GNE, encoding the allosteric site of UDPGlcNAc 2-epimerase/ManNAc kinase, mutated in sialuria patients
(Seppala et al., 1999), as well as the coding regions of Neu5Ac
pyruvate lyase (NPL, Neu5Ac aldolase) (Wu et al., 2005), and
CMP-Neu5Ac synthase (CMAS) (Lawrence et al., 2001), did not
reveal any mutation either.
Clinical characteristics of five patients
with isolated elevation of free sialic
acid in CSF
Two out of the five patients (Patients 1 and 2) were siblings but
with no reported consanguinity. All patients presented with progressive cerebellar ataxia that started during early adulthood
except in patient 5 (Table 1). Cognitive and/or noticeable behavioural decline started concomitantly (Table 1). A peripheral neuropathy was also found in all patients on clinical and/or
electrophysiological examination (Table 1). Three patients manifested non-neurological symptoms such as bifascicular block, QT
interval increase and glomerulosclerosis (Table 1).
Brain MRI revealed a mild to moderate vermian atrophy in all
patients (Fig. 2A). White matter abnormalities were observed
in the cerebellum (n = 4), particularly in the hilus of the dentate
nucleus and peridentate white matter (Fig. 2B), and in the brainstem (n = 2). White matter signal abnormality was also seen at the
supratentorial level, involving the periventricular white matter and
sparing the juxtacortical U fibres (n = 3) (Fig. 2C and D). This
abnormality extended to the pyramidal tracts in the thalamus
and to the basal ganglia in two patients (Fig. 2C). Note that,
apart from mild vermian atrophy, one of the two affected sisters
presented with almost normal brain imaging.
Due to the complex neurological presentation, a muscle biopsy
was performed in three patients (Patients 1, 4 and 5 from Table 1)
with immunohistochemistry and enzymatic studies that showed no
abnormality. Sequencing of genes commonly involved in cerebellar
ataxia (SCA 1-2-3-6-7-14-17, FRDA, AO1, AO2, DRPLA, mitochondrial DNA) was negative in all five patients. In addition,
Patient 4 had no mutation in the POLG1 gene (Milone et al.,
2008).
F. Mochel et al.
Hyposialylation of CSF transferrin of
the five CAFSA patients
In order to determine whether the elevation of CSF free sialic acid
could reflect functional changes in the metabolism of free sialic
acid, we studied the patterns of sialylation of an abundant CSF
protein, transferrin. Proteomic studies were performed on the five
CAFSA patients, as well as on 15 age-matched disease controls
from the cohort previously described. All CAFSA patients displayed
elevated total protein in the CSF (range 54–1.72 mg/dl). Using
two-dimensional gel electrophoresis followed by protein and
glycoforms analysis using MS and MS/MS, a difference in the
neuraminic acid isoforms of one of the most abundant CSF
proteins, transferrin, was identified (Fig. 3A). When compared to
age-matched disease controls and Salla patients, affected patients
had a greater ratio of asialotransferrin (not containing sialic acid)
to total transferrin (40 7.7 versus 24.9 6.4 and 15 8.7)
(Fig. 3B). The asialotransferrin/total transferrin ratio in CAFSA
patients was significantly elevated compared to this ratio in disease
controls (P50.001) and also compared to Salla disease patients
(P = 0.003). There was no significant difference between disease
controls and Salla patients.
Investigation of peripheral nerve in two
patients
Light and electronic microscopy of the sural nerve biopsy of
Patient 3 showed mild loss of large myelinated fibres and hypomyelination of small and regenerating fibres. No other abnormality
was seen (data not shown). Similar findings were seen in the nerve
biopsy of Patient 5 with the addition of marked polylobulation of
Schwann cell nuclei (data not shown).
Oligosaccharides are key components of nerve gangliosides, and
require the transfer of free sialic acid molecules by sialyltransferases. Therefore, we analysed the profile of nerve gangliosides
in order to better characterize the biochemical defects in CAFSA
patients. Ganglioside profiles studied in peripheral nerve biopsies
of Patients 3 and 5 did not reveal any qualitative abnormality
compared to control nerves. No abnormality was either detected
in a patient with Salla disease. In all cases, 30 LM1 was the major
ganglioside entity, and the proportion of major di- and trisialosialogangliosides was unchanged (Supplementary figure). The total
concentration of gangliosides could not be measured accurately
due to the small size of the biopsies, but appeared normal from
visual inspection of the chromatograms.
Discussion
We describe a novel neurometabolic entity involving free sialic
acid in five patients with cerebellar ataxia as the leading symptom,
named CAFSA (cerebellar ataxia with free sialic acid). The five
patients had an elevation of free sialic acid in CSF but not in
urine, plasma or in cultured skin fibroblasts. This new entity
emphasizes the original contribution of NMRS of CSF in the investigation of neurological disorders of unknown aetiology. Increased
asialotransferrin relative to total transferrin was also found in the
Ataxia and free sialic acid
Table 1 Clinical characteristic of five CAFSA patients. Patients 1 and 2 are siblings
Patient 2
Patient 3
Patient 4
Patient 5
Sex
Age of onset (ataxia) (years)
Age at examination (years)
Family history
Cerebellar gait ataxia
Cerebellar dysarthria
Eye movements
Tendon reflexes UL
Tendon reflexes LL
Plantar reflexes
Peripheral nerve electrophysiology
Female
24
34
Yes
+++
++
Slow saccades
+2
+1
Flexor
Sensory axonal neuropathy
Female
20
37
Yes
++
+
Slow saccades
+1
0
Flexor
ND
Male
22
23
No
+++
+
Normal
+4
+1
Flexor
Motor axonal neuropathy
Male
20
35
No
+++
+
Slow saccades
+2
+2
Flexor
Motor axonal neuropathy
Conduction velocities; Amplitudes
(normal values)
Ulnar Sensory; amplitude
ND
Peroneal Motor
Peroneal Motor
Female
10
19
No
+++
++
Slow saccades
0
0
Extensor
Sensory and motor
demyelinating neuropathy
Peroneal Motor
Paranoid episodes
45 m/s (442 m/s); 0.4 mV
(43 mV)
Marked mental slowness
32 m/s (445 m/s);
0.5 mV (43.4 mV)
Frequent anger outbursts
_
_
_
_
_
+++
_
Growth retardation
Ptosis/external ophthalmoplegia
+++
Mixed retinal dystrophy
Bifascicular block
Cataract
5.7 mV (48 mV)
Cognition/Behavioral decline
Other
Hearing loss
Vision loss
Non-neurological
Features
Frontal syndrome
(attention deficit)
Dystonia/Epilepsy
_
_
_
UL = upper limbs; LL = lower limbs; + (mild), ++ (moderate), +++ (severe); – (not present).
21 m/s (440 m/s);
2.1 mV (42.4 mV)
Low range IQ (88),
attention deficit
_
+
_
Growth retardation
Long QT interval
Glomerulosclerosis
Brain 2009: 132; 801–809
Patient 1
| 805
806
| Brain 2009: 132; 801–809
Figure 2 Brain MRI of CAFSA Patients 1, 3 and 5. For each
patient, (A) T1-weighted mid-sagittal view (arrow points to
cerebellar atrophy); B–D: axial T2-weighted images at the level
of the posterior fossa (B) basal ganglia (C) and centrum semi
ovale (D). Arrows point to white matter abnormalities, and
arrowhead to the involvement of the basal ganglia in Patient 5.
CSF of all CAFSA patients, suggesting that the sialylation of key
central nervous system proteins is altered. This prompted us to
investigate gangliosides in the peripheral nerve but we found no
evidence of abnormal sialylation of sphingolipids in this tissue.
In addition to cerebellar ataxia, all patients had peripheral neuropathy and cognitive decline or important behavioural changes.
A mild to moderate cerebellar atrophy restricted to the vermis was
observed in all CAFSA patients, often associated with supra- and
infratentorial white matter abnormalities, especially around the
dentate nucleus. The peridentate white matter hyperintense signal
on T2-weighted images is similar to the one seen in cerebrotendinous xanthomatosis where it is due to the accumulation of
cholestanol (Sedel et al., 2008) and in some mitochondrial diseases.
These diseases could be considered as differential diagnoses. Three
out of the four patients tested presented with axonal peripheral
neuropathy. Confounding factors such as post kidney-transplant
type 2 diabetes and multiple medications may have modified the
neuropathy of Patient 5. Yet, the pathological findings on the sural
nerve biopsy of Patient 5 were similar to those of Patient 3.
Based on this combination of clinical and imaging characteristics
we prospectively identified a sixth patient suspected of having
CAFSA. A 44-year-old male was diagnosed with deafness as a
child and developed cerebellar ataxia at the age of 40 years,
together with cognitive decline. His brain MRI was quite similar
to the pattern described in the CAFSA patients, i.e. moderate
cerebellar atrophy and white matter abnormalities both at the
F. Mochel et al.
Figure 3 Proteomic studies in the CSF of CAFSA patients.
(A) Two-dimensional gel electrophoresis of CSF transferrin with
identification of sialic acid-containing isoforms versus nonsialic acidcontaining isoforms confirmed in all patient groups by MALDI-TOF
TOF as previously described (Vanderver et al., 2005). (B) Ratio of
asialotransferrin to total transferrin showing a significant difference
between CAFSA patients (n = 5) and disease controls (n = 15),
as well as between CAFSA and Salla patients (n = 3).
Figure 4 Free sialic acid metabolism and transport. See the
Discussion for details. Numbers 1–5 designate the metabolic
steps that we investigated in the five CAFSA patients, with
steps 1, 2, 4 and 5 indicating the four candidate genes
sequenced in the free sialic acid biosynthetic pathway that did
not reveal any mutation.
periventricular level and in the region of the dentate nucleus.
NMRS confirmed a marked elevation of free sialic acid in his
CSF (66 mmol/l). Despite unifying clinical characteristics, the intrafamilial and extrafamilial phenotypic heterogeneity observed in the
Ataxia and free sialic acid
Brain 2009: 132; 801–809
CAFSA patients is compatible with either genetic homogeneity with
variable expression or genetic heterogeneity. The occurrence of
the disease in two siblings with unaffected parents suggests an
autosomal recessive transmission.
The first two committed steps of cytoplasmic free Neu5Ac
synthesis is mediated by the bifunctional enzyme UDP-GlcNAc
2-epimerase/ManNAc kinase (encoded by the GNE gene). The
epimerase enzymatic domain converts UDP-GlcNAc to N-acetylmannosamine (ManNAc) and the kinase domain subsequently
phosphorylates ManNAc to ManNAc-6-P. The epimerase domain
is feed-back inhibited by CMP-sialic acid in its allosteric site
(encoded by exon 5 of GNE, Fig. 4, Step 1) (Hinderlich et al.,
1997; Seppala et al., 1999). ManNAc-6-P is sequentially further
converted to Neu5Ac. Neu5Ac is then translocated to the nucleus,
where it is activated to CMP-Neu5Ac, by the CMAS (Fig. 4, Step
2). After exiting the nucleus, CMP-Neu5Ac can either cytoplasmically inhibit UDP-GlcNAc 2-epimerase activity, or be transported
into the Golgi where various sialyltransferases utilize CMP-Neu5Ac
to sialylate oligosaccharides that participate in the synthesis of
glycoproteins and gangliosides (Fig. 4, Step 3) (Varki, 1997;
Keppler et al., 1999). For recycling, glycoproteins and gangliosides
enter the lysosome, where free Neu5Ac is cleaved from the sialyloligosaccharides by neuraminidase and is then exported out of the
lysosome by SCL17A5 (Fig. 4, Step 4) (Verheijen et al., 1999).
Neu5Ac pyruvate lyase (Neu5Ac aldolase) finally catalyzes the
cleavage of Neu5Ac into pyruvate and ManNAc (Fig. 4, Step 5).
It has recently been suggested that Neu5Ac can also be taken up
from an exogenous source through macropinocytosis and incorporated into different subcellular fractions (Fig. 4) (Bardor et al.,
2005). Free sialic acid levels can be measured in human body
fluids such as urine, plasma or CSF. Urinary excretion of free
sialic acid is increased in two disorders associated so far with
| 807
sialic acid metabolism (Strehle, 2003), the SASD—due to mutations in SLC17A5, and sialuria—caused by mutations in the
allosteric site of GNE (Seppala et al., 1999). To appear in urine
or serum, free sialic has to exit the cell, but such mechanisms
have not been described. The cellular exit of free sialic acid
could involve exocytosis or a membrane transporter (Fig. 4).
In order to identify possible aetiologies of CAFSA syndrome, we
investigated several metabolic and genetic aspects of free sialic
acid metabolism. The association of increased cerebrospinal sialic
acid and hyposialylation of CSF proteins has never been described,
especially in the context of absence of intracellular accumulation
of free sialic acid. Hereditary inclusion body myopathy (HIBM) is
due to mutations in the GNE gene resulting in reduced activity of
both the UDP-GlcNAc 2-epimerase and the ManNAc kinase
enzymes. HIBM is associated with hyposialylation of -dystroglycan, an integral component of the dystrophin–glycoprotein complex, in HIBM muscle (Huizing et al., 2004; Saito et al., 2004) but
normal sialylation profile in serum (Savelkoul et al., 2006).
Likewise, we hypothesized that our patients may display mutations
in one or more genes of the free sialic acid pathway, possibly
resulting in hyposialylation of brain proteins, such as transferrin.
We therefore excluded mutations in the SLC17A5 gene of the five
patients, at the genomic and mRNA levels. No mutations in exon
5 of GNE, and in the coding regions of the genes for CMPNeu5Ac synthase and Neu5Ac pyruvate lyase were found
(Fig. 4). Since the free sialic acid elevation appears to be restricted
to the CSF, there may exist unreported—exclusively neuronal
expressed—alternative transcripts of some of these genes.
Table 2 shows the comparison of the main features of CAFSA
with the known free sialic disorders.
Our results may also suggest that, instead of intracellular, there
may be an abnormal trafficking of free sialic acid between the
Table 2 Main features of CAFSA compared with known free sialic acid disorders
Age at onset
Horizontal nystagmus
Cerebellar ataxia
Pyramidal syndrome
Cognitive abnormalities
Peripheral neuropathy
Dysmorphism
Growth retardation
Signs of organ storage
Cerebral MRI
Cerebellar atrophy
Thin corpus callosum
White matter abnormality
Free sialic acid elevation
Urine
CSF
Fibroblasts
CAFSA
Salla disease
(Aula and Gahl, 2001)
ISSD
(Lemyre et al., 1999)
Sialuria
(Aula and Gahl, 2001)
10–24 years
No
Yes
No
Cognitive or behavioural
decline
Axonal4demyelinating
No
Possible
No
Infancy to Childhood
Common
Yes
Yes
Psychomotor retardation
50% (demyelinating)
Mild
Yes
No
1st year of life
Yes
Yes
ND
Psychomotor
retardation (severe)
ND
Yes
Yes
Yes
Infancy
No
No
No
Psychomotor
retardation (mild)
ND
Yes
No
Yes
Mild to moderate
No
Hilus of dentate
nucleus, peridentate
white matter and
periventricular
Moderate to severe
Yes
Diffuse hypomyelination
Severe
Yes
Diffuse hypomyelination
No
No
No
No
Yes
No
Common(+)
Yes
Yes (lysosomal)
Yes (++)
ND
Yes (lysosomal)
Yes (+++)
ND
Yes (cytoplasmic)
Salla patients have been reported without sialuria (Mochel et al., Ann Neurol, in press). ISSD = Infantile free sialic storage disease; ND = not determined; + = mild;
++ = moderate; +++ = massive.
808
| Brain 2009: 132; 801–809
intracellular and the extracellular compartments. The marked elevation of this sugar in the CSF, even above the levels seen in Salla
disease, a disorder with intracellular accumulation of free sialic
acid, gives some support to this hypothesis. Other research
approaches, such as the analysis of bio-orthogonal reactions to
monitor the transport and metabolism of sialylated biomolecules
in patients’ cell lines (Yarema and Bertozzi, 2001; Prescher and
Bertozzi, 2005) may be required to further elucidate this new neurological disorder of free sialic acid metabolism.
Supplementary material
Supplementary material is available at Brain online.
Acknowledgements
We would like to thank Drs Odile Dubourg, Maria Tsokos, Mones
Abu-Asab and Kondi Wong for the pathological analysis of the
patients’ sural nerve, Dr Roseline Froissart and Nathan H. McNeill
for their contribution in SLC17A5 sequencing and Dr Jerry N.
Thompson for the measurements of free sialic acid in patients’
urine. The authors are also grateful to Nadège Boildieu, Hakima
Manseur and Sylvie Forlani for their assistance with patients’
samples.
Funding
Assistance Publique des Hôpitaux de Paris (CRC 05169);
Intramural Program of the National Institute of Neurological
Disorders and Stroke; National Human Genome Research
Institute; National Institute of Health; Baylor Research
Foundation. Integrated Molecular Core for Rehabilitation
Medicine (NIH IDDRC P30HD40677, NIH NCMRR/NINDS 5R24
HD050846).
References
Aula P, Gahl WA. Disorders of free sialic acid storage. In: Valle D,
Beaudet AL, Vogelstein B, Kinzler KW, Antonarakis SE, Balabio A,
editors. The online metabolic & molecular bases of inherited disease.
New York: McGraw-Hill; 2001. p. 1–30.
Bardor M, Nguyen DH, Diaz S, Varki A. Mechanism of uptake and
incorporation of the non-human sialic acid N-glycolylneuraminic acid
into human cells. J Biol Chem 2005; 280: 4228–37.
Engelke UF, Liebrand-van Sambeek ML, de Jong JG, Leroy JG, Morava E,
Smeitink JA, et al. N-acetylated metabolites in urine: proton nuclear
magnetic resonance spectroscopic study on patients with inborn errors
of metabolism. Clin Chem 2004; 50: 58–66.
Engelke UF, Sass JO, Van Coster RN, Gerlo E, Olbrich H, Krywawych S,
et al. NMR spectroscopy of aminoacylase 1 deficiency, a novel inborn
error of metabolism. NMR Biomed 2008; 21: 138–47.
Hayakawa K, De Felice C, Watanabe T, Tanaka T, Iinuma K, Nihei K,
et al. Determination of free N-acetylneuraminic acid in human body
fluids by high-performance liquid chromatography with fluorimetric
detection. J Chromatogr 1993; 620: 25–31.
Hinderlich S, Stasche R, Zeitler R, Reutter W. A bifunctional
enzyme catalyzes the first two steps in N-acetylneuraminic acid
F. Mochel et al.
biosynthesis of rat liver. Purification and characterization of
UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase.
J Biol Chem 1997; 272: 24313–8.
Huizing M, Rakocevic G, Sparks SE, Mamali I, Shatunov A, Goldfarb L, et al.
Hypoglycosylation of alpha-dystroglycan in patients with hereditary IBM
due to GNE mutations. Mol Genet Metab 2004; 81: 196–202.
Keppler OT, Hinderlich S, Langner J, Schwartz-Albiez R, Reutter W,
Pawlita M. UDP-GlcNAc 2-epimerase: a regulator of cell surface sialylation. Science 1999; 284: 1372–6.
Kleta R, Aughton DJ, Rivkin MJ, Huizing M, Strovel E, Anikster Y, et al.
Biochemical and molecular analyses of infantile free sialic acid storage
disease in North American children. Am J Med Genet A 2003; 120A:
28–33.
Lawrence SM, Huddleston KA, Tomiya N, Nguyen N, Lee YC, Vann WF,
et al. Cloning and expression of human sialic acid pathway genes to
generate CMP-sialic acids in insect cells. Glycoconj J 2001; 18:
205–13.
Lemyre E, Russo P, Melancon SB, Gagne R, Potier M, Lambert M.
Clinical spectrum of infantile free sialic acid storage disease. Am J
Med Genet 1999; 82: 385–91.
Milone M, Brunetti-Pierri N, Tang LY, Kumar N, Mezei MM, Josephs K,
et al. Sensory ataxic neuropathy with ophthalmoparesis caused by
POLG mutations. Neuromuscul Disord 2008; 18: 626–32.
Mochel F, Barritault J, Boldieu N, Eugene M, Sedel F, Durr A, et al.
Contribution of in vitro NMR spectroscopy to metabolic and neurodegenerative disorders. Rev Neurol 2007; 163: 960–5.
Moolenaar SH, Engelke UF, Wevers RA. Proton nuclear magnetic resonance spectroscopy of body fluids in the field of inborn errors of
metabolism. Ann Clin Biochem 2003; 40: 16–24.
Oostendorp M, Engelke UF, Willemsen MA, Wevers RA. Diagnosing
inborn errors of lipid metabolism with proton nuclear magnetic resonance spectroscopy. Clin Chem 2006; 52: 1395–405.
Prescher JA, Bertozzi CR. Chemistry in living systems. Nat Chem Biol
2005; 1: 13–21.
Romppanen J, Mononen I. Age-related reference values for urinary
excretion of sialic acid and deoxysialic acid: application to
diagnosis of storage disorders of free sialic acid. Clin Chem 1995;
41: 544–7.
Saito F, Tomimitsu H, Arai K, Nakai S, Kanda T, Shimizu T, et al. A
Japanese patient with distal myopathy with rimmed vacuoles: missense
mutations in the epimerase domain of the UDP-N-acetylglucosamine
2-epimerase/N-acetylmannosamine kinase (GNE) gene accompanied
by hyposialylation of skeletal muscle glycoproteins. Neuromuscul
Disord 2004; 14: 158–61.
Savelkoul PJ, Manoli I, Sparks SE, Ciccone C, Gahl WA, Krasnewich DM,
et al. Normal sialylation of serum N-linked and O-GalNAc-linked
glycans in hereditary inclusion-body myopathy. Mol Genet Metab
2006; 88: 389–90.
Sedel F, Tourbah A, Fontaine B, Lubetzki C, Baumann N, Saudubray JM,
et al. Leukoencephalopathies associated with inborn errors of metabolism in adults. J Inherit Metab Dis 2008; 31: 295–307.
Seppala R, Lehto VP, Gahl WA. Mutations in the human UDP-N-acetylglucosamine 2-epimerase gene define the disease sialuria and the
allosteric site of the enzyme. Am J Hum Genet 1999; 64: 1563–9.
Strehle EM. Sialic acid storage disease and related disorders. Genet Test
2003; 7: 113–21.
Timmons M, Tsokos M, Asab MA, Seminara SB, Zirzow GC, Kaneski CR,
et al. Peripheral and central hypomyelination with hypogonadotropic
hypogonadism and hypodontia. Neurology 2006; 67: 2066–9.
Vanderver A, Hathout Y, Maletkovic J, Gordon ES, Mintz M,
Timmons M, et al. Sensitivity and specificity of decreased CSF
asialotransferrin for eIF2B-related disorder. Neurology 2008; 70:
2226–32.
Vanderver A, Schiffmann R, Timmons M, Kellersberger KA, Fabris D,
Hoffman EP, et al. Decreased asialotransferrin in cerebrospinal fluid
of patients with childhood-onset ataxia and central nervous system
hypomyelination/vanishing white matter disease. Clin Chem 2005;
51: 2031–42.
Ataxia and free sialic acid
Varki A. Sialic acids as ligands in recognition phenomena. Faseb J 1997;
11: 248–55.
Verheijen FW, Verbeek E, Aula N, Beerens CE, Havelaar AC, Joosse M,
et al. A new gene, encoding an anion transporter, is mutated in sialic
acid storage diseases. Nat Genet 1999; 23: 462–5.
Brain 2009: 132; 801–809
| 809
Wu M, Gu S, Xu J, Zou X, Zheng H, Jin Z, et al. A novel splice variant of
human gene NPL, mainly expressed in human liver, kidney and peripheral blood leukocyte. DNA Seq 2005; 16: 137–42.
Yarema KJ, Bertozzi CR. Characterizing glycosylation pathways. Genome
Biol 2001; 2: REVIEWS0004.