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doi:10.1093/brain/awh401 Brain (2005), 128, 711–722 Neuropathological, biochemical and molecular findings in a glutaric acidemia type 1 cohort Christopher B. R. Funk, 1,5 Asuri N. Prasad, 6 Patrick Frosk, 3 Sven Sauer, 7 Stefan Ko ¨lker, 7 Cheryl R. Greenberg 3,4 and Marc R. Del Bigio 1,2,5 Correspondence to: Marc R. Del Bigio MD PhD FRCPC, Canada Research Chair in Developmental Neuropathology, Department of Pathology, University of Manitoba, D212-770 Bannatyne Avenue, Winnipeg MB, R3E 0W3, Canada E-mail: [email protected] Departments of 1 Pathology, 2 Human Anatomy and Cell Science, 3 Biochemistry and Medical Genetics and 4 Pediatrics and Child Health, Faculty of Medicine, University of Manitoba, 5 Manitoba Institute of Child Health, Winnipeg, 6 Department of Pediatrics, University of Western Ontario, London, Ontario, Canada and 7 Department of General Pediatrics, Division of Metabolic and Endocrine Diseases, University Children’s Hospital, Heidelberg, Germany. Summary Glutaric acidemia type 1 (GA-1) is an autosomal recessive disorder characterized by a deficiency of glutaryl-CoA dehydrogenase (GCDH) activity. GA-1 is often associated with an acute encephalopathy between 6 and 18 months of age that causes striatal damage resulting in a severe dys- tonic movement disorder. Ten autopsy cases have been pre- viously described. Our goal is to understand the disorder better so that treatments can be designed. Therefore, we present the neuropathological features of six additional cases (8 months – 40 years), all North American aboriginals with the identical homozygous mutation. This cohort dis- plays similar pathological characteristics to those previ- ously described. Four had macroencephaly. All had striatal atrophy with severe loss of medium-sized neurons. We present several novel findings. This natural time course study allows us to conclude that neuron loss occurs shortly after the encephalopathical crisis and does not progress. In addition, we demonstrate mild loss of large striatal neur- ons, spongiform changes restricted to brainstem white matter and a mild lymphocytic infiltrate in the early stages. Reverse transcriptase-PCR to detect the GCDH mRNA revealed normal and truncated transcripts similar to those in fibroblasts. All brain regions demonstrated markedly elevated concentrations of GA (3770–21 200 nmol/g pro- tein) and 3-OH-GA (280–740 nmol/g protein), with no evid- ence of striatal specificity or age dependency. The role of organic acids as toxic agents and as osmolytes is discussed. The pathogenesis of selective neuronal loss cannot be explained on the basis of regional genetic and/or metabolic differences. A suitable animal model for GA-1 is needed. Keywords: autopsy; glutaric acid; 3-hydroxyglutaric acid; striatum; molecular genetics Abbreviations: ChAT = choline acetyltransferase; DAB = diaminobenzidine; H&E = haematoxylin and eosin; GA = glutaric acid; GA-1 = glutaric acidemia type 1; GCDH = glutaryl-CoA dehydrogenase; GFAP = glial fibrillary acidic protein; HLA-DR = human leucocyte antigen-DR; NMDA = N-methyl-D-aspartate; 3-OH-GA = 3 hydroxyglutaric acid; RT-PCR = Reverse transcription polymerase chain reaction Received July 14, 2004. Revised December 10, 2004. Accepted December 13, 2004. Advance Access publication February 2, 2005 Introduction Glutaric acidemia type 1 (GA-1) is an autosomal recessive disorder of amino acid metabolism caused by the deficiency of functional glutaryl-CoA dehydrogenase (GCDH) activity (Christensen, 1993), an essential enzyme in the catabolic path- ways of L-tryptophan, L-lysine and L-hydroxylysine (Goodman et al., 1977; Baric et al., 1998; Goodman and Frerman, 2001). Lack of functional GCDH activity typically leads to the accumulation of glutaric acid (GA), 3-hydroxyglutaric acid (3-OH-GA) and glutaconic acid in the blood, urine, CSF and brain tissue (Stokke et al., 1975; Goodman et al., 1977; Baric et al., 1998). GA and 3-OH-GA might induce an imbalance in glutamatergic and GABAergic neurotransmission (Wajner et al., 2004) and 3-OH-GA might act through excitotoxic NMDA receptors to produce a neurotoxic effect (Ko ¨lker # The Author (2005). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: [email protected] Downloaded from https://academic.oup.com/brain/article/128/4/711/284316 by guest on 10 February 2022
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Page 1: Neuropathological, biochemical and molecular findings in a glutaric

doi:10.1093/brain/awh401 Brain (2005), 128, 711–722

Neuropathological, biochemical and molecularfindings in a glutaric acidemia type 1 cohort

Christopher B. R. Funk,1,5 Asuri N. Prasad,6 Patrick Frosk,3 Sven Sauer,7 Stefan Kolker,7

Cheryl R. Greenberg3,4 and Marc R. Del Bigio1,2,5

Correspondence to: Marc R. Del Bigio MD PhD FRCPC,

Canada Research Chair in Developmental Neuropathology,

Department of Pathology, University of Manitoba,

D212-770 Bannatyne Avenue, Winnipeg MB,

R3E 0W3, Canada

E-mail: [email protected]

Departments of 1Pathology, 2Human Anatomy and Cell

Science, 3Biochemistry and Medical Genetics and4Pediatrics and Child Health, Faculty of Medicine,

University of Manitoba, 5Manitoba Institute of Child

Health, Winnipeg, 6Department of Pediatrics, University

of Western Ontario, London, Ontario, Canada and7Department of General Pediatrics, Division of Metabolic

and Endocrine Diseases, University Children’s Hospital,

Heidelberg, Germany.

SummaryGlutaric acidemia type 1 (GA-1) is an autosomal recessive

disorder characterized by a deficiency of glutaryl-CoA

dehydrogenase (GCDH) activity. GA-1 is often associated

with an acute encephalopathy between 6 and 18 months

of age that causes striatal damage resulting in a severe dys-

tonicmovement disorder. Tenautopsy cases havebeenpre-viously described. Our goal is to understand the disorder

better so that treatments can be designed. Therefore, we

present the neuropathological features of six additional

cases (8months – 40 years), all NorthAmerican aboriginals

with the identical homozygous mutation. This cohort dis-

plays similar pathological characteristics to those previ-

ously described. Four had macroencephaly. All had

striatal atrophy with severe loss of medium-sized neurons.Wepresent several novel findings. This natural time course

study allows us to conclude that neuron loss occurs shortly

after the encephalopathical crisis and does not progress.

In addition, we demonstratemild loss of large striatal neur-

ons, spongiform changes restricted to brainstem white

matter and amild lymphocytic infiltrate in the early stages.

Reverse transcriptase-PCR to detect the GCDH mRNArevealed normal and truncated transcripts similar to those

in fibroblasts. All brain regions demonstrated markedly

elevated concentrations of GA (3770–21 200 nmol/g pro-

tein) and3-OH-GA (280–740nmol/g protein),with no evid-

ence of striatal specificity or age dependency. The role of

organic acids as toxic agents and as osmolytes is discussed.

The pathogenesis of selective neuronal loss cannot be

explained on the basis of regional genetic and/or metabolicdifferences. A suitable animal model for GA-1 is needed.

Keywords: autopsy; glutaric acid; 3-hydroxyglutaric acid; striatum; molecular genetics

Abbreviations: ChAT = choline acetyltransferase; DAB = diaminobenzidine; H&E = haematoxylin and eosin; GA = glutaric

acid; GA-1 = glutaric acidemia type 1; GCDH = glutaryl-CoA dehydrogenase; GFAP = glial fibrillary acidic protein;

HLA-DR = human leucocyte antigen-DR; NMDA = N-methyl-D-aspartate; 3-OH-GA = 3 hydroxyglutaric acid;

RT-PCR = Reverse transcription polymerase chain reaction

Received July 14, 2004. Revised December 10, 2004. Accepted December 13, 2004. Advance Access publication

February 2, 2005

IntroductionGlutaric acidemia type 1 (GA-1) is an autosomal recessive

disorder of amino acid metabolism caused by the deficiency

of functional glutaryl-CoA dehydrogenase (GCDH) activity

(Christensen, 1993), an essential enzyme in the catabolic path-

ways of L-tryptophan, L-lysine and L-hydroxylysine (Goodman

et al., 1977; Baric et al., 1998; Goodman and Frerman, 2001).

Lack of functional GCDH activity typically leads to the

accumulation of glutaric acid (GA), 3-hydroxyglutaric acid

(3-OH-GA) and glutaconic acid in the blood, urine, CSF and

brain tissue (Stokke et al., 1975; Goodman et al., 1977; Baric

et al., 1998). GA and 3-OH-GA might induce an imbalance in

glutamatergic and GABAergic neurotransmission (Wajner

et al., 2004) and 3-OH-GA might act through excitotoxic

NMDA receptors to produce a neurotoxic effect (Kolker

# The Author (2005). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: [email protected]

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et al., 2004a,b), although this is not supported by all experi-

mental data (Freudenberg et al., 2004; Lund et al., 2004). In

one autopsy case, 3-OH-GA was relatively more abundant in

the striatum (Kolker et al., 2003), where the neuronal damage

is most severe.

GA-1 affected children are clinically characterized by

macrocephaly appearing at, or shortly after, birth and initial

normal development interrupted by abrupt onset of dystonia

and choreoathetosis, which then remain relatively static.

Neurological abnormalities usually appear between 6 and

18 months of age, often in conjunction with a febrile illness.

Intellect seems to be relatively preserved (Goodman and

Frerman, 2001). The profound neurological sequelae may

lead to death in early childhood; however, some individuals

survive for many years. A minority of biochemically affected

individuals may remain asymptomatic or experience an insi-

dious onset of mild neurological abnormalities. The brains of

children affected with GA-1 exhibit wide Sylvian fissures and

enlarged frontal ventricles due to caudate atrophy. There are

only 10 published autopsy reports of GA-1 (Goodman et al.,

1977;Leibeletal., 1980;Bennettetal., 1986;Chowetal.,1988;

Bergman et al., 1989; Soffer et al., 1992; Kimura et al., 1994;

Kolker et al., 2003). Atrophy and severe neuronal loss affecting

the caudate and putamen are always present. Spongiform

change in the white matter has been frequently described.

This disease is over-represented among North American

aboriginals (Ojibway-Cree) in a genetic isolate in north-

eastern Manitoba and north-western Ontario in central

Canada (Haworth et al., 1991). In this population, the carrier

frequency is �1 in 10. Twenty-eight affected children, many

the products of consanguineous unions, have been identified

since 1970; 21 individuals have suffered an encephalopath-

ical crisis with severe striatal damage (unpublished data).

Although the phenotype is severe, the amount of GA and

3-OH-GA in blood and urine tends to be very low (Haworth

et al., 1991). All affected individuals are homozygous for a

splicing mutation, a G to T transversion at the +5 position

of intron 1 in the gene encoding GCDH (IVS-1 +5 G>T)

(Greenberg et al., 1995; Goodman et al., 1998). This splicing

mutation allows for some normal splicing with a transcript of

the expected size, as well as a truncated transcript resulting

from activation of a cryptic splice site 26 base pairs (bp)

upstream in exon 1; the cryptic splicing leads to a frame

shift and premature termination.

There are many gaps in the present understanding of the

neuropathogenesis of striatal injury in this disorder. Here we

describe the neuropathological findings in the brains of five

children and one adult with GA-1, all with the same mutation.

This is of particular interest because the range of survival times

and the availability of frozen tissue for genetic analysis

might offer additional insight into pathogenesis of the disorder.

Material and methodsThis was a retrospective neuropathological study of six individuals

of North American aboriginal background diagnosed with GA-1.

All patients in this study were examined and diagnosed by clinical

geneticists at the Children’s Hospital/Health Sciences Centre in

Winnipeg, Canada. There have been 13 known deaths in this cohort

between 1978 and 2004; complete autopsies have been performed on

six cases. The age of death ranged from 8 months to 40 years. In all

cases, the medical records were reviewed in detail. Original brain

imaging studies were available for review from only three cases. For

each case, histological samples from one or two anonymized control

cases with no neurological disease and matched for age and gender

were obtained from the autopsy archives. Frozen control tissues were

more limited and not as closely comparable. This study was con-

ducted with approval of the University of Monitoba Biomedical

Research Ethics Board as well as the Pathology Access Committee

for Tissue.

Archived paraffin blocks, glass slides and hospital records were

retrieved for all cases. The brains had been reasonably well sampled

with 8–20 tissue blocks per brain available for microscopic exam-

ination. All slides were examined by one neuropathologist (M.R.D.).

Age- and sex-matched controls were identified and similar levels of

the striatum were sampled for each case. Sections from striatal

blocks were stained with haematoxylin and eosin (H&E). Immuno-

histochemical staining was performed to detect glial fibrillary acidic

protein (GFAP) (polyclonal anti-GFAP; 1/1200 dilution; DakoCyto-

mation (Carpinteria CA, USA), activated microglia (anti-HLA-DR;

1/250 dilution; Dako), lymphocytes (anti-CD3; 1/100 dilution;

Dako) and synaptic vesicle protein synaptophysin (1/25 dilution;

Dako). Neurons were identified with the use of anti-NeuN (neuronal

nuclei) (1/800; Chemicon International). To identify inhibitory

interneurons, antibodies to calbindin (1/100 dilution; Chemicon)

and g-aminobutyrate (anti-GABA; 1/125; Chemicon) were used.

To identify noradrenergic axons, anti-tyrosine hydroxylase (dilution

1/75; Chemicon) was used. Choline acetyltransferase (ChAT)

(anti-ChAT; 1/250 dilution; Chemicon) Chemicon International

(Temecula CA, USA) was used to identify large cholinergic neurons.

GFAP, human leucocyte antigen-DR (HLA-DR), CD3 and synapto-

physin antibodies were detected using the Envision detection sys-

tem. GABA, ChAT, NeuN and tyrosine hydroxylase antibodies were

detected with biotinylated secondary antibodies, streptavidin horse-

radish peroxidase and 3,30- diaminobenzidine (DAB). A fluorescent

secondary antibody (Cy-3) was used with the calbindin primary

antibody. Appropriate negative controls were used in all cases.

Neuron counts were performed on H&E stained sections. This was

done because the neurons have a fairly characteristic morphology

and because we found the immunostaining to be inconsistent in the

autopsy material. Counts were made in the dorsal and ventral regions

of both the caudate and putamen at an ocular magnification of 4003.

The size of the ocular reticule counting square was 250mm 3 250mm.

The counts consisted of neurons contained within six adjacent focal

areas in each of the four regions stated above. Only neurons that

could be unambiguously identified based on cytological details were

counted. National Institutes of Heath (NIH) image analysis software

was used to measure the density of DAB precipitation—as an indic-

ator of the magnitude of immunoreactivity—with antibodies against

GFAP, HLA-DR and synaptophysin. Images used for NIH analysis

were taken in the dorsal and ventral regions of the caudate and

putamen at 103 objective magnification. Two images were obtained

from each region; their densities were then averaged to give a better

representation of immunoreactivity in each area. Neuron counts and

immunohistochemical labelling data were tested for normal distribu-

tion. Paired t-tests were then used to compare differences between

cases and age-matched controls using StatView 5 Software (SAS;

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Page 3: Neuropathological, biochemical and molecular findings in a glutaric

Cary, NC, USA). Regression analysis was used to test for age-

dependent effects.

Reverse transcription PCR (RT-PCR) was performed on total

RNA isolated from frozen brain tissue stored at �70�C following

autopsy of four cases. The method has been previously described for

analysis of fibroblasts and lymphoblasts from this cohort (Greenberg

et al., 1995). Briefly, following reverse transcription of RNA from

homogenized tissue, two overlapping fragments of the complete

GCDH cDNA were generated by separate standard PCR reactions.

PCR conditions were 2 ml cDNA in 50 ml of 50 pmol of each primer,

200 mM of each dNTP, 5 ml of 103 PCR reaction buffer (Perkin

Elmer, Boston, MA, USA) and 1ml AmpliTaq (8 units) (Applied

Biosystems, Foster City CA, USA) for 35 cycles at 95�C/3 min,

95�C for 1 min, 58�C for 1 min, and 72�C for 1 min with a final 10

min cycle at 72�C. Products were analysed on 6% acrylamide mini-

gels with ethidium bromide (10 mg/ml) staining. RT-PCR for

glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used

as a positive confirmation of mRNA integrity and isolation. Similar

analyses were performed on brain tissues obtained from non-age

matched patients (because the supply of control material was lim-

ited) without known neurological disease and with similar post-

mortem delay. All analyses were done blinded.

Analysis of organic acid (GA and 3-OH-GA) concentrations was

performed on frozen brain tissue from four GA-1 and three control

cases. The samples were shipped by courier on dry ice to Germany.

The analyses were performed in a blinded manner. The methods for

this analysis are described in detail in previous studies (Schor et al.,

2002; Kolker et al., 2003). Again due to limited supply, the controls

were not age matched.

ResultsThe ages of the individuals with GA-1 ranged from 8 months

to 40 years. Body weights and heights, GA levels in urine

(Seargeant et al., 1992) and GCDH activity are shown in

Table 1. In the youngest four cases, the brains were much

larger than expected for age. A description of clinical and

neuropathological findings follows in order of ascending age.

Case 1This male had six out of seven older siblings also affected by

GA-1. He is a member of pedigree B described in the clinical

report of this cohort (Haworth et al., 1991). At full term birth

in 1993, he was said to be jittery. A brief seizure occurred at

3 months. Developmental delay, mild hypotonia and head

bobbing were noted at 4 months. At 6 months, a CT scan

showed slightly enlarged ventricles, temporal fossa fluid

collections and mild widening of frontal sulci. The head

circumference was on the 99.9 percentile at birth, 2 months

and 8 months. At 8 months, he presented with vomiting,

diarrhoea, fever and severe dehydration. He became rapidly

Table 1 Clinical and autopsy data of patients with glutaric acidemia

Case # 1 2 3 4 5 6

Gender Male Male Male Male Female MaleAge at neurologicalcrisis

6.5 months 10.5 months 7 months 5.5 months 6 months 4 months

Age at death 8 months 12 months 16 months 18.5 months 7 years 7 months 40 yearsHeight/length(percentile)

73 cm (50) 75 cm (25) 79 cm (25) 74 cm (<5) 114 cm (5) Not known

Body weight(percentile)

9.4 kg (75) 9.1 kg (15) 7.7 kg (<5) 8.1 kg (<5) 21 kg (25) 43 kg (<5)

Brain weight(deviation fromexpected weight)

1320 g >+9 SD 980 g >+2 SD 1176 g >+2 SD 1104 g +1 SD 1300 g >median 1635 g >median

GCDH mutationpresent

+ / + Not done + / + + / + + / + + / +

GCDH lymphocyteenzyme activity(% of normal)

Not done Not done 4 % Not done 9 % 7 %

Urine GA(mmol/mmolcreatinine)(normal < 10)y

33–450 Not done 58 99–118 6–97 13

Urine 3-OH-GA(mmol/mmolcreatinine)

Small amount Not done Small amount Slight increase Trace

Delay in autopsy 20 h 11 h 48 h 18 h 40 h 5.5 hFrozen braintissue sampled

Cerebellum None Caudate,cerebellum,frontalcerebrum

Frontalcerebrum

Striatum,frontalcerebrum

None

yNone of these individuals had GA or 3-OH-GA assayed in blood or CSF. Other members of the cohort who were tested had normal orundetectable levels.

Neuropathology in GA-1 713

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comatose. A CT scan showed haemorrhage in the right tem-

poroparietal region and he died 3 days later. Autopsy revealed

widespread ischaemic neuronal damage in the cerebrum and

cerebellum. Haemorrhagic infarction in the right parietal and

temporal cerebrum was due to venous sinus thrombosis,

apparently a complication of sepsis and dehydration. Mild

symmetric dilation of the lateral ventricles accompanied

atrophy of the striatum. There was severe loss of medium-

sized neurons in the caudate and putamen, with dorsal areas

more severely affected than ventral. Immunohistochemical

stains demonstrated astrocyte hypertrophy and microglial

activation in the striatum. There were rare scattered and a

few perivascular clusters of CD3 immunoreactive lymphocytes

in the putamen. No white matter vacuoles were identified.

Case 2Following full term birth in 1977, this male sibling of Case 1

had mild respiratory distress requiring incubation. He is

the presumed first GA-1 case of pedigree B described in

the clinical report of this cohort (Haworth et al., 1991). At

10 months of age, acute bacterial (E.coli) pneumonia was

accompanied by fever and lethargy. Cerebrospinal protein

was elevated. Despite antibiotic treatment, the fever contin-

ued and 3 weeks later he developed episodes of opisthotonus,

limb stiffening and lethargy. A CT scan was reported to show

enlarged lateral ventricles and Sylvian fissures. On Pheno-

barbital, there was a slight improvement of his neurological

status. He suffered respiratory arrest following aspiration of

vomit �6 weeks after presentation. Autopsy revealed pneu-

monia. The head circumference was on the 71.9 percentile.

The external surface of the brain appeared normal, but the

ventricles were mildly enlarged. The caudate and putamen

exhibited widespread loss of medium-size neurons with

marked astrocytic proliferation, microglial activation and

focal dystrophic calcification. Mild diffuse lymphocyte infilt-

ration and rare perivascular cuffing (up to 5 cells thick) was

present in the putamen. The tail of the caudate adjacent to the

hippocampus appeared normal. Rare vacuoles were seen in

the white matter of a single gyrus and vacuoles were fairly

abundant in the central tegmental tract of the brainstem, but

not elsewhere.

Case 3This male’s parents were known heterozygotes for the GA-1

mutation. He is Case 12 in pedigree C described in the clinical

report of this cohort (Haworth et al., 1991). Following full-

term birth in 1989, he presented at 7 months of age with

developmental delay and relatively sudden onset of dystonia

and seizures. A CT scan showed hypointensity of the caud-

ate and putamen, and widening of the Sylvian fissures. He

became severely impaired, was treated with Phenobarbital

and required placement of a feeding tube. He was placed in

a chronic care institution. At 16 months, he developed fever

and died suddenly. Autopsy showed acute glottitis and pneu-

monitis. His head circumference was on the 22 percentile.

The external appearance of the brain was unremarkable. The

caudate and putamen were atrophic and the lateral ventricles

were mildly enlarged. Microscopically, the striatum exhibited

loss of medium-size neurons, plump reactive astrocytes,

reactive microglia, scattered calcospherites and rare CD3

immunoreactive lymphocytes in the caudate. The tail of the

caudate adjacent to the hippocampus appeared normal. There

were scattered pyknotic neurons in the cerebral cortex.

Case 4This male was born at 39 weeks by Caesarean section and was

found, on genetic screening in 1999, to have GA-1 (he is

Case 3 in Greenberg et al., 2002). His development was

delayed slightly. At 5.5 months of age, he developed fever

with onset of dystonia and athetoid limb movements as well

as seizure activity. A CT scan showed enlarged frontal horns

of the lateral ventricles and widened Sylvian fissures, but no

generalized atrophy (Fig. 1). Caudate atrophy was worse

at 10 months. He was treated with Phenobarbital and

Fig. 1 Photographs showing a CT scan of the head in thehorizontal plane obtained 8 months before death (upper) and acoronal slice of the brain (lower) from Case 4. Both exhibitflattening of the head of the caudate nucleus along the wall of thelateral ventricle (arrows) and mild enlargement of the lateralventricles. Note that the cortical thickness is essentially normal.

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topiramate, but failed to thrive and had multiple respiratory

infections. At 15 months, he was unable to sit but had some

head control and visual interaction. During a febrile illness at

18 months, he stopped breathing. Autopsy revealed laryngitis

and dehydration. His head circumference had been on the

52 percentile at birth, 89 percentile at 3 months, 63 percentile

at 8 months and was on the 50 percentile at the time of death.

The brain exhibited mild widening of temporal and frontal

sulci, and pronounced widening of the Sylvian fissures. No

histological abnormalities were apparent in the cerebral cor-

tex of the temporal lobe tips or frontal lobes. The caudate

nuclei were small, yellowish and firm. There was symmetric

lateral ventricle enlargement (Fig. 1). The head of the caudate

and the putamen exhibited severe neuronal loss with pro-

nounced reactive astrocytes. Only rare reactive microglia

were identified. The tail of the caudate adjacent to the hip-

pocampus appeared normal. The cerebral white matter exhib-

ited no vacuoles, no damaged axons were identified using

amyloid precursor protein immunohistochemistry, and no

myelin debris could be demonstrated with the Marchi

method. Rare vacuoles were present in the central tegmental

tract at the level of the midbrain.

Case 5This female born by forceps delivery at 37 weeks gestation,

in 1983, had generalized hypotonia in infancy. She is Case 1

in pedigree A described in the clinical report of this cohort

(Haworth et al., 1991). At 6 months of age, she developed

seizures and choreoathetoid movements. Thereafter she

developed severe spastic quadriparesis. She was unable to

walk and developed severe scoliosis. A CT scan at 5 years

showed enlarged frontal horns and slightly wide Sylvian

fissures. She died of acute pneumonia at age 7 years. Her

head circumference had been on the 25 percentile at birth,

52 percentile at 6 months, 3 percentile at 19 months and

25 percentile at the time of death. Her brain exhibited mild

gyral flattening, slightly enlarged ventricles and severe striatal

atrophy. The caudate and putamen had a near complete loss of

medium size neurons (Fig. 2) and some GFAP immunoreactive

astrocytes; the nucleus accumbens was spared. Scattered

hypertrophic astrocytes and very rare HLA-DR immuno-

reactive microglia were present in the internal capsule—

although there were no vacuoles. There were small foci of

spongiform change in the frontal and insular cortex.

Case 6This male born at 40 weeks gestation, in 1953, was apparently

slow to breathe. He had three siblings that died in infancy

from pneumonia. He is Case 14 in pedigree E described in

the clinical report of this cohort (Haworth et al., 1991). At

12 months of age, he developed a high fever and was admitted

to hospital with dystonia. At 6 years, he was documented

to have severe flexor spasticity of the arms and legs,

choreoathetoid movements and oral dyskinesia. Seizures

were reported only early in life. He resided in a nursing

care facility for his entire life. At no time was brain imaging

conducted and head circumference information was not

available. He required many hospitalizations for aspiration

pneumonia. On one such admission at age 40 years, the

treatment was complicated by pseudomembranous colitis

from which he died. His brain had a normal external appear-

ance. The frontal horns of the lateral ventricles were moder-

ately enlarged. There was marked atrophy of the caudate and

putamen with neuronal loss and abundant corpora amylacea.

There was no significant immunoreactivity for GFAP or

HLA-DR. The anterior nucleus accumbens was spared.

The white matter of the cerebrum and brainstem was well

myelinated and no vacuoles were identified. Subtle chronic

astrogliosis, demonstrable with modified phosphotungstic

acid haematoxylin (PTAH) stain, was evident in the inferior

olivary nuclei but there was no obvious neuron loss. Very rare

empty basket cells were present in the cerebellum, suggestive

of mild Purkinje cell loss. Frontal cortex tissue was examined

in all cases, none provided evidence of obvious cell loss or

reactive changes.

Comparative analysisQuantitative comparison of the striatum in GA 1 cases and

controls showed statistically significant (P < 0.05) loss of

Fig. 2 Photomicrographs of the striatum showing (A) normalneuron density (neurons indicated by arrows) in an age-matchedcontrol and (B) severe loss of neurons in the same area of GA-1Case 5. (H&E stained sections, 403 objective magnification,bar = 20 mm).

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medium sized neurons in the dorsal caudate, ventral caudate,

dorsalputamenandventralputamen(Fig.3).Thedorsal regions

of the caudate and putamen were more severely affected,

although this was not statistically significant. There was no

age-dependent trend (Fig. 4) in the quantity of neurons in

the GA-1 cases, suggesting that maximal neuron loss had

occurred within 2 months of onset of symptoms, which was

the time of death after encephalopathical crisis in Case 1. Large

neurons, which are normally much less abundant than the

medium-sized neurons, were significantly fewer in the ventral

putamen of the GA1 patients with similar trends in all areas of

the striatum (Fig. 5). Immunostaining for GFAP (Figs 6 and 7)

demonstrated the presence of reactive astrocytes in all areas of

the striatum, with a tendency to greater staining in the dorsal

striatum. Analysis of reactive microglial activation (Fig. 8)

demonstrated significant HLA-DR immunoreactivity in

only the three youngest cases and mild infiltrate in the fourth

case, suggesting that it only persists a few months after the

acute episode. Overall, the differences approached statistical

significance (P = 0.0725 and 0.0699, respectively; paired t-test

versus age-matched controls) only in the dorsal and ventral

putamen.

Loss of GABA and calbindin immunoreactivity confirmed

that the population of medium sized neurons is decreased in

GA-1 (data not shown). The relative absence of NeuN label-

ling confirmed that neurons were lost and not simply atrophic.

Qualitative inspection of ChAT and tyrosine hydroxylase

immunoreactivity in large cholinergic and dopaminergic

neurons indicated little, if any, difference between GA-1

Fig. 3 Counts of medium sized neurons (mean 6 SE) in fourregions of the striatum. A significant loss of medium sized neuronswas seen in dorsal and ventral areas of the caudate and putamen;paired t-test versus age-matched controls, *P < 0.008.

Fig. 6 Photomicrographs showing scant immunoreactivityfor GFAP in dorsal caudate of a control case (A) and abundantGFAP-positive reactive astrocytes in GA-1 case 1(B). (DAB detection of anti-GFAP with haematoxylincounterstain; bar = 50 mm).

Fig. 4 Neuron density as a function of age. Note that themagnitude of neuron loss is similar regardless of the age of theGA-1 patient.

Fig. 5 Counts of large neurons (mean 6 SE) in the striatum.There was a marginally significant loss in the ventral caudate;paired t-test versus age-matched controls, P = 0.0422.

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cases and controls (data not shown). Synaptophysin immun-

oreactivity in striatum was not significantly different between

cases and controls (data not shown), indicating the preserva-

tion of input axons to the striatum.

The presence of GAPDH amplification product in both

GA-1 cases and controls indicated the presence of undegraded

mRNA despite long post-mortem delays to autopsy and the

lengthy interval between autopsies and molecular study.

However, there was limited frozen tissue stored and striatal

tissue was not recoverable. Thus, proper quantification of the

relative proportion of normal and mutant GCDH transcripts

was not possible. Nonetheless, we observed abundant mutant

and normal sized GCDH transcripts in the frontal cortex of

Cases 3 and 5 (the most abundant frozen tissue available)

(Fig. 9). A similar pattern has been seen in fibroblasts and

lymphoblasts of affected patients (Greenberg et al., 1995).

Organic acid analysis demonstrated marked elevations of

GA and 3-OH-GA compared with controls. There was no

evidence of striatal specificity or age dependency. There

was a slight elevation of GA in one control brain, which

likely can be considered a non-specific change related to

agonal events (Table 2).

Fig. 7 Proportionate area with DAB precipitate (measured by NIHimage analysis; mean 6 SE) when labelled with primary antibodyagainst GFAP; paired t-test versus age-matched controls*P < 0.016, **P < 0.005.

Fig. 8 Photomicrographs showing HLA-DR immunolabellingof reactive microglia in the putamen. The age matched controlcase (A) exhibits no cells while the GA-1 sample (Case 1, B)exhibits abundant activated cells. (DAB detection of anti-HLA-DR with haematoxylin counterstain, bar = 50 mm).

Fig. 9 PCR amplification products from frontal lobe samplesincubated without (–RT) and with reverse transcriptase ( + RT),respectively, are shown from Case 3 (lanes 1 and 2), Case 5 (lanes3 and 4), Control 1 (lanes 5 and 6) and Control 2 (lanes 7 and 8).The upper band in lanes 2, 4, 6 and 8 represents a normal sizedfragment of 393 bp and the lower band in lanes 2 and 4 representsthe truncated message 26 bp shorter than the normalsized-fragment. Lane 9 is the l00 bp ladder.

Table 2 Analysis of organic acids in brain tissue samplesfrom four GA-1 and three control cases

Brain sample areas Age atdeath

GA(nmol/gprotein)

3-OH-GA(nmol/gprotein)

Case 1 – cerebellum 8 months 21 240 740Case 3 – cerebellum 16 months 8020 550

– thalamus 10 960 360– caudate 5960 360– frontal 7760 280

Case 4 – frontal 18 months 3770 360Case 5 – frontal 7 years

7 months5990 290

– caudate/internal capsule

8310 420

Control – frontal 3 weeks n.d. n.d.Control – frontal 6 months n.d. n.d.Control – frontal 5 months 170 n.d.

n.d.= not detected (i.e. <10).

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DiscussionThe neuropathological changes associated with GA-1

described here in six individuals are essentially similar to

those previously described in 10 affected children of other

ethnic backgrounds ranging in age from 10 months to

15 years (Goodman et al., 1977; Leibel et al., 1980; Bennett

et al., 1986; Chow et al., 1988; Bergman et al., 1989; Soffer

et al., 1992; Kimura et al., 1994; Kolker et al., 2003). Atrophy

involving the striatum is consistently described, with sparing

of the caudate in one case (Kimura et al., 1994). We observed

sparing of the nucleus accumbens and tail of the caudate

nucleus. In seven out of ten previously published cases,

spongiform change of the white matter was observed. An

additional autopsy by Kolker and colleagues (unpublished)

on a female aged 3 years and 3 months, with disease onset

at 15 months, demonstrated severe neuron loss and gliosis in

the basal nuclei, widespread moderate neuron loss in the

cerebral cortex, brainstem (including substantia nigra) and

cerebellum and diffuse spongiform changes in white matter.

There was minimal spongiform white matter change in the

cases described here and it was largely confined to the brain-

stem. Whether this reflects a cohort difference or simply a

timing difference is not clear. We did not find clear evidence

of axon damage or demyelination in any case, the exception

being subtle microglial activation in the internal capsule of

Case 5. This suggests that the white matter changes could

represent a fluid shift into myelin rather than a destructive

change. The anatomical restriction in these cases cannot

readily be explained. Imaging studies suggest that white matter

alterations are rare in the Amish cohort (Strauss et al., 2003).

However, inaboywith GCDHdeficiency presenting at15years

age without major neurological abnormality, magnetic reson-

ance spectroscopy suggested increased myelin turnover in

conjunction with a reduction of neuronal integrity (Bodamer

et al., 2004). In contrast, a 19-month old child studied by the

same technique exhibited changes suggestive of neuroaxonal

damage and demyelination (Kurul et al., 2004). Obviously, the

only way to truly understand such studies is to perform them on

individuals who shortly thereafter undergo autopsy.

We have previously reported both normally spliced and

deleted cDNA in fibroblasts and lymphoblasts of our affected

patients (Greenberg et al., 1995) and we now observe similar

findings in brain tissue. Thus, there does not appear to be a

direct correlation between the presence of wild type GCDH

transcript in the regions of the brains available for study and

clinical outcome. Unfortunately, cDNA could not be prepared

from the regions of the basal nuclei to assess expression

in these vulnerable areas of the brain. Nonetheless, it seems

reasonable to assume that GCDH intron 1 splicing is probably

comparable in these areas of the brain. Our results suggest

that splicing alone is probably not the sole determinant of

clinical outcome. It is possible that, in the target regions,

aberrantly spliced mRNA is unstable or is prematurely

degraded; other genetic factors influencing higher order

RNA structure or RNA protein interactions are likely to affect

the final clinical outcome.

Biochemically, the most intriguing result of our study

is the discrepancy between high concentrations of GA and

3-OH-GA in brain and low concentrations in urine and plasma

(Haworth et al., 1991). The concentrations are much higher

than in one previously described patient who was treated with

a lysine-restricted diet (Kolker et al., 2003). However, GA

concentrations are comparable with those found in a post-

mortem examination of another published case (Goodman

et al., 1977) and in a frontal cortex biopsy of an untreated

adult who excreted high levels of GA (S. Kolker, unpublished

data). We failed to confirm the prior observation that con-

centrations of GA and 3-OH-GA are highest in the striatum

(Kolker et al., 2003). The high gradient of organic acids

towards the brain and the low permeability of the blood–

brain barrier for dicarboxylic acids in general (Hoffmann

et al., 1993) support the notion that organic acids are probably

generated in the brain. However, this has not yet been expli-

citly proven for GA or 3-OH-GA, and requires detailed

further investigation, including permeability studies of the

blood–brain barrier and blood–CSF barrier. Unfortunately,

we do not have data concerning CSF concentrations of GA

and 3-OH-GA in this cohort of patients (Haworth et al., 1991;

Greenberg et al., 2002).

Despite genetic homogeneity and a wide range in duration

of survival following encephalopathical crisis, all cases had

near complete loss of medium neurons from the striatum,

with the exception of the nucleus accumbens and tail of the

caudate. This likely occurs within, at most, a few weeks of the

first encephalopathical crisis. It is important to emphasize

the apparent lack of progression over the lifespan because

this observation supports the idea that a single severe insult

during infancy creates the bulk of striatal injury. The three

individuals that died soonest after the encephalopathical crisis

(Cases 1, 2 and 3) had small collections of lymphocytes in the

cerebrum. This is not a common incidental finding in child-

hood death; it might simply reflect a septic state rather than a

specific component of GA-1 brain damage or it could be

interpreted as evidence that mild encephalitis can precipitate

the striatal destruction. Reactive microglia, which can

accompany neuron loss or encephalitis, also dissipated within

6 months of the encephalopathical crisis. Because the neuro-

toxic effect of 3-ON-GA per se is weak (Freudenberg et al.,

2004; Lund et al., 2004), it has been suggested that additional

amplifying mechanisms are necessary to initiate neuronal

damage. Among these, induction of nitric oxide, synthase

and indoleamine 2,3-oxygenase by inflammatory cytokines

have been considered as relevant, increasing the formation of

nitric oxide and quinolinic acid, respectively. The occurrence

of infections in early life may provide the trigger for a cas-

cade of injurious events. Reactive astrocytes, which are activ-

ated acutely perhaps as a protective response (Porciuncula

et al., 2004), persist many years post injury.

Quantification of large cholinergic neurons has never been

performed in cases of GA-1; these cells have been reported as

unaffected. If this population of neurons was undamaged, one

would demonstrate an increased density in atrophied striatum

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whose medium neurons had been lost. Our results indicate

that there is a significant loss of large neurons from some

regions of the striatum. They receive glutamatergic input

from the thalamus that is mediated by NMDA, AMPA and

mGluR receptors (Haber and Gdowski, 2004) and therefore

should be vulnerable. Quinolinic acid has been shown to

damage cholinergic neurons (Rossato et al., 2002; Guidetti

and Schwarcz, 2003; Kumar, 2004). The observed dorso-

ventral gradient of neuron loss might be explained in different

ways. First, the neuron characteristics as well as the afferent

and efferent connections to the striatum exhibit dorso-ventral

and rostro-caudal differences (Haber and Gdowski, 2004),

which are likely to influence vulnerability. Secondly, blood

flow through lenticulostriate arteries could, in the face of the

excitotoxic stress/hyperactivity, be diverted away from the

dorsal regions leading to a additive hypoxic/ischaemic injury.

There are other conditions with overlapping neuropatho-

logical features including familial infantile striatal necrosis

(Straussberg et al., 2002), Huntington disease (Portera-

Cailliau et al., 1995), neuroacanthosis and Wilson disease

(Nelson, 1995), most of which have a gradual progressive

course. The presentation of the acute encephalopathical crisis

in GA1 resembles that of a ‘metabolic stroke’ (i.e. acute

neuronal damage induced by toxic agents), in contrast to

vascular occlusion or hypoxia/ischaemia as in peripartum

neonatal encephalopathy (Johnston, 2001). The result is a

rapid selective loss of vulnerable neurons rather than an

infarct. Our results support the prior assertion that GA-1

preferentially targets striatal medium-spiny neurons, whereas

cholinergic neurons are affected to a lesser extent. The mech-

anisms underlying this particular vulnerability have been the

subject of much debate and have been well investigated in

models for adult neurodegenerative disorders (Bates, 2003)

and for a few paediatric neurological disorders (hypoxia

ischemia in the term infant, bilirubin encephalopathy); they

may well share final overlapping pathways. Several variables

should be considered in a hypothesis to explain the selective

striatal damage. A neuro-anatomical basis might play a role.

The striatum receives strong glutamatergic corticostriatal and

thalamostriatal input as well as dopaminergic input (Haber

and Gdowski, 2004). Within the striatum, GABAergic

medium-spiny neurons are more vulnerable than cholinergic

aspiny interneurons to energy compromise (Nishino et al.,

2000). Specific membrane ion channels, glutamate receptor

subtypes and subunits, and intracellular enzymatic activities

are involved in the events responsible for differential vulner-

abilities to oxygen or glucose deprivation and to glutamate

receptor-mediated toxicity (Calabresi et al., 2000). Suscept-

ibility to nitric oxide mediated cell damage also strongly

differs among striatal neurons, with medium-spiny neurons

being the most vulnerable (Dawson, 1995). Quinolinic acid

production via shunting through the kynurenine pathway was

postulated to play a role in GA-1 some years ago (Heyes,

1987) and has been reiterated as a mechanism through which

intercurrent infection could precipitate damage (Varadkar

and Surtees, 2004). Recent pathophysiological models for

GA-1 refer to many of the above mentioned aspects, hypo-

thesizing a role for accumulating organic acids acting via

direct or indirect overactivation of glutamatergic receptors

(in particular NMDAr) resulting in increased influx of

calcium and increased generation of reactive oxygen species

(Kolker et al., 2004a,b). Notably, concentrations of 3-OH-

GA in the present post-mortem CNS investigations are at the

same level as the lowest concentrations of 3-OH-GA that

cause neuronal damage in vitro (Kolker et al., 2004a).

Alternate mechanisms have been also considered. Strauss

and Morton (2003) speculated on alternative mechanisms

of organic acids, such as damage to the microvasculature

with modulation of blood–brain barrier permeability, altera-

tion of astrocyte function (Frizzo et al., 2004; Muhlhausen

et al., 2004). Integrity of the blood–brain barrier and

functional status of astrocytes cannot be assessed by our

methods; there is no morphological evidence for changes

in the vasculature.

The timing of injury needs to be considered; most children

who suffer an encephalopathical crisis do so by �1 year of

age and rarely if ever after 5 years (Goodman and Frerman,

2001; Strauss et al., 2003). Some of the vulnerability might be

explained on the basis of postnatal developmental changes.

Several studies have examined the concentration of glutama-

tergic receptors in human forebrain using 3H-glutamate and3H-MK801 binding assays. In the cerebral cortex, the con-

centration appears to be relatively low in newborns and peaks

sometime between 5 months and 1–2 years, thereafter declin-

ing. Changes in the modulatory sites of the NMDA receptor

can also be demonstrated by pharmacological studies

(Kornhuber et al., 1988, 1989; D’Souza et al., 1992; Piggott

et al., 1992; Slater et al., 1993; Chahal et al., 1998). The

quantity of aspartate binding sites also peaks at around

5 months postnatal in the cortex; this peak in glutamatergic

synapses could be related to plasticity (Slater et al., 1992).

Fewer studies have directly examined the human striatum; the

quantity of glutamate binding sites is roughly similar in puta-

men and frontal cortex (Kornhuber et al., 1988) and the

concentrations of glutamate and aspartate increase rapidly

during the first postnatal year (Kornhuber et al., 1993). Stud-

ies in the postnatal rat during the first month of life, an age

that roughly corresponds to human infancy, have defined at

the molecular level the shifts that occur in NMDA receptor

subtypes (Monyer et al., 1994; Portera-Cailliau et al., 1996;

Gurd et al., 2002). Particular NMDA receptor subtypes may

make neurons especially vulnerable in infancy (McQuillen

and Ferriero, 2004). Additionally and in relation to the

infectious aspect discussed above, the onset of a vulnerable

period could reflect the loss of passive immunity after which

damage is initiated by exposure to, presumably ubiquitous,

infectious agents.

The gross brain findings also need to be addressed. The

temporal fossa fluid collections have been interpreted by

some as an indicator of atrophy. However, at least two

patients in this cohort (not described in this report) identified

by newborn screening had the abnormality at 1 and 6 weeks

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age, without significant neurological impairment. This has also

been described by others (Neumaier-Probst et al., 2004). The

one case where we were able to examine the temporal lobe tip

exhibited no obvious abnormalities and there were no insular

cortex abnormalities in any case. Therefore, this represents a

developmental abnormality, i.e. temporal hypotrophy or arach-

noid cyst-like anomaly, rather than atrophy. The absence of

generalized subarachnoid space enlargement suggests that this

is not a disorder of CSF absorption, as has been suggested

previously (Martinez-Lage, 1996). Concerning the macroen-

cephaly and megalencephaly, only two out of six individuals

had enlarged heads during infancy, while brain weight deviated

substantially from the median only in the three youngest cases

(up to 16 months). In a clinical follow-up study of Nordic

patients, the head circumference relative to normal values

peaks at 6 months of age and tends to normalize thereafter

(Kyllerman et al., 2004). Together, the observations suggest

that the brain is most enlarged around the time that encepha-

lopathical crises occur. If the organic acids act as osmotic

agents, brain enlargement could partly be accounted for largely

by water accumulation; however, our observation that concen-

tration of the organic acids is not correlated with age to some

extent negates the idea. Alternative mechanisms, such as a

role for GA or 3-OH-GA as osmoregulators (in analogy to

N-acetylaspartate in aspartoacylase deficiency) have not yet

been investigated (Baslow, 2003). Because our data do not

indicate that the concentrations decrease with age, ‘normal-

ization’ of head size and brain weight might be accounted

for by mild atrophy beyond the striatum, which can be almost

impossible to detect histologically.

With the advent of screening among vulnerable popula-

tions, GA-1 can now be identified presymptomatically at

birth. Current treatments for newborns identified presympto-

matically with GA-1 involve dietary modifications and very

aggressive treatment of intercurrent infections (Strauss et al.,

2003; Naughten et al., 2004). Outcome in some children

treated presymptomatically appears to be improved, but

acute brain injury and its devastating sequelae still occur

in others (Greenberg et al., 2002). Our evidence of a single

insult and data indicating that excitatory synapses peak in the

first year of life opens up the possibility that presymptomatic

detection and the use of neuroprotective agents could be tried

to limit brain injury. Anti-inflammatory agents might also be

of value. Whether preservation of neurons (e.g. with a phar-

maceutical agent) in infancy could allow them to mature into

a less vulnerable phenotype is not known. An animal model

of GA-1 that mimics the human neuropathology is required to

test possible treatment interventions. However, a useful

animal model is not yet available (Funk et al., 2004; Koeller

et al., 2004). Further comparative clinical studies of individu-

als with GA1 in this cohort who do not suffer an encephalo-

pathical crisis could provide some insight. For example, if

GA levels in the CSF simply correlated with enzyme activity

and with clinical severity, a dose-response relationship could

be invoked. However, when considering all mutations, there

is no simple correlation between genotype and phenotype;

there is some evidence that low excretors tend to be more

impaired (Christensen et al., 2004). The significance of this

finding is not yet known. One might speculate that low

urinary excretion of organic acids in some patients reflects

high tissue retention. Alternately, accumulating organic acids

might influence the expression of relevant proteins, such as

neurotransmitter receptors, thereby altering the susceptibility

to neuronal damage—analogous to chemical preconditioning

(Riepe et al., 1997; Ravati et al., 2001; Kolker et al., 2002).

More work and a representative animal model are required

to fully understand the pathogenesis of this disorder

(Goodman, 2004).

AcknowledgementsWe wish to thank members of the Winnipeg GA-1 study

group and especially Louise Dilling for helping to identify

patients. We wish to thank Melissa Caswill for help with

review of the records and Sharon Allen, Susan Janeczko,

Patrick Feyh and Christy Pylypjuk for technical assistance.

This work was funded by grants to M.D., A.P. and C.R.G.

from the Garrod Association of Canada and the Manitoba

Medical Service Foundation and a grant to S.K. from

the German Research Community (DFG KO 2010/2-1).

Dr Del Bigio holds the Canada Research Chair in Develop-

mental Neuropathology.

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