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J Inherit Metab Dis (2010) 33:411419 DOI 10.

1007/s10545-010-9134-2

ORIGINAL ARTICLE

Fumaric aciduria: an overview and the first Brazilian case report


Gabriella Allegri & Marcia J. Fernandes & Fernanda B. Scalco & Patricia Correia & Ruth E. Simoni & Juan C. Llerena Jr & Maria L. Costa de Oliveira

Received: 18 January 2010 / Revised: 5 May 2010 / Accepted: 12 May 2010 / Published online: 15 June 2010 # SSIEM and Springer 2010

Abstract Fumaric aciduria is a rare metabolic disease, with 40 cases reported so far. Fumarase deficiency leads mainly to brain abnormalities, developmental delay, and great accumulation of fumaric acid in urine. This work presents the first case of fumaric aciduria described in Brazil, which presented with some interesting clinical and biochemical findings such as colpocephaly, hepatic alterations, and marked metabolic acidosis since birth. Common findings were ventriculomegaly, hypotonia, and microcephaly. Biochemically, besides the high urinary fumaric acid excretion, atypical elevation of plasma citrulline, tyrosine and methionine levels were also observed. In order to show all features and variants of fumaric aciduria, literature data of 40 patients was reviewed and compared with the case reported here. Findings in all these patients demonstrate that this disorder does not yet have its phenotype completely defined; it is important that more patients be described.

Communicated by: Jan Smeitink Competing interest: None declared. G. Allegri (*) : M. J. Fernandes : F. B. Scalco : R. E. Simoni : M. L. C. de Oliveira Laboratrio de Erros Inatos do Metabolismo (LABEIM), Departamento de Bioqumica, Instituto de Qumica, Universidade Federal do Rio de Janeiro (UFRJ), Cidade Universitria, Ilha do Fundo, Centro de Tecnologia, bloco A, 536 C, 21941 900 Rio de Janeiro, Brasil e-mail: gabiallegri@yahoo.com.br P. Correia : J. C. Llerena Jr Centro de Gentica Mdica, Instituto Fernandes Figueira, FIOCRUZ, Rio de Janeiro, Brasil

Abbreviations AICAR 5-aminoimidazole-4-carboxamide ribotide AMP adenosine monophosphate AS argininosuccinase ASL adenylosuccinate lyase ASS argininosuccinic synthetase AST aspartate aminotransferase CPSI carbamoylphosphate synthetase I. FAH fumarylacetoacetate hydrolase FAICAR 5-formaminoimidazole-4-carboxamide ribotide FH fumarate hydratase GD glutamate dehydrogenase 4-HPPD 4-hydroxy-phenylpyruvate dioxygenase IMP inosine monophosphate MAI maleylacetoacetate isomerase ME malic enzyme MDH malate dehydrogenase OT ornithine transcarbamoylase PC pyruvate carboxylase PDHC pyruvate dehydrogenase complex SAICAR 5-aminoimidazole-(N-succinylcarboxiamide) ribotide SDH succinate dehydrogenase TAT tyrosine aminotransferase

Introduction Fumaric aciduria, an autosomal recessive disorder, is caused by deficiency of the enzyme fumarate hydratase [fumarase (FH)]. The first patient with fumarase deficiency (OMIM 136850), presenting with lethargy, microcephaly, and hypotonia combined with high urinary fumaric acid excretion, was described by Zinn et al. (1986). Fumaric aciduria, however, had already been reported in 1983 by

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Whelan et al in a couple of siblings (Whelan et al 1983). Fumaric aciduria is characterized by massive excretion of fumaric acid in urine. Other metabolites may also be altered in body fluids: Krebs cycle intermediates, other dicarboxylic acids (suberic, adipic), and succinylpurine derivatives. The disease is considered rare, since in the last 27 years, only 40 cases have been reported in the literature. However, a higher frequency can be observed at the border of northern Arizona and southern Utah, USA. This is probably due to a founder effect in closed, religious polygamist communities living in these regions, among which marriage between relatives seems to be frequent (Kerrigan et al 2000, Szep 2007). Fumaric aciduria is classified into the group of disorders that involve metabolic energy. In this group are included mitochondrial disorders that may interfere in embryophetal development, leading to developmental delay, seizures, motor dysfunction, and alterations in other organ systems, especially liver, kidney, and blood cells (Saudubray et al 2006; Filiano 2006). FH (E.C. 4.2.1.2) catalyzes the stereospecific and reversible hydration/dehydration of fumarate to S-malate. This enzyme is homotetrameric, iron-dependent, and thermostable. It exists in two isoforms, mitochondrial and cytosolic, both being encoded by the same gene located on chromosome 1q.42.1 (Estevez et al 2002, Remes et al 2004). They only differ at the aminoterminal residue: pyroglutamic acid on the mitochondrial isoform and Nacetylalanine on the cytosolic one (Akiba et al 1984). In fumarase deficiency, both isoforms are affected. It is supposed that the cytosolic isoform processes fumaric acid from the urea cycle, amino acid, and purine metabolism, whereas the mitochondrial isoform originates from the Krebs cycle (Lehtonen 2006). The aim of this study was to report the first case of fumaric aciduria in Brazil and to review data from patients found in the literature.

Case report A girl, the fourth child of consanguineous parents (first cousins), was born at 38 weeks of gestation after a pregnancy complicated by polyhydramnios. Birth weight was 2180 g (< 3rd percentile), length 47 cm (10th percentile), head circumference 30 cm (< 3rd percentile), and APGAR score 8/9. One brother, born at term, and one premature sister, had both died 6 h(presumed perinatal asphyxia) and two days (cause not reported), respectively, after birth. Ultrasound images of the fourth child showed agenesis of the corpus callosum and mild bilateral pyelocaliceal dilatation. At physical examination, dysmorphic features were noted, such as hypertelorism, overriding

sutures, hooked nose, micrognathia, camptodactyly of the fifth finger, single transverse palmar crease on the left hand and postaxial polydactyly on the right hand. Immediately after birth and during the following 17 days, she presented with hypoglycemic episodes difficult to control (glucose: 2040 mg/dl). Laboratory exams, performed during one of these episodes, showed lactic acidemia (4.0 mol/ml; N: 0.72.5 mol/ml), mild hyperammonemia, and marked metabolic acidosis (pH 7.20; pCO2 25.1 mmHg; HCO39.6 mEq/L; BE -17.5 mEq/L). Neurologic exam, performed at 4 days of life, showed incomplete Moro reflex, posture of limbs flexion, reduced axial tonus, and slightly elevated appendicular tonus. At 6 days of life, antibiotics were administered for one week due to suspicion of sepsis. On the seventh and 25th days, two samples of urine and one of plasma were collected and sent for inborn errors of metabolism (IEM) screening. Amino acid analyses were carried out on a Biochrom 20 (Biochrom Ltd., Cambridge, UK) amino acid analyzer. Sample preparation and analysis were performed according to the manufacturers standard protocols. General aminoaciduria was observed and some plasma amino acids were also altered (Table 1). Urinary organic acid analyses by gas chromatography mass spectrometry (GCMS) (according to Tanaka et al 1980) showed massive excretion of fumaric acid (> 232 mmol/ mol creatinine, normal range < 45 mmol/mol creatinine). The same result was presented by a new urine sample collected at 40 days of life. At 1 month of age, despite breast feeding and normal sucking, the baby had extreme difficulty gaining weight, remaining undernourished due to persistent vomiting and occasional diarrhea. Cholestatic jaundice and hepatomegaly were noted since birth. At 40 days of life, the following hepatic enzymes were altered: [aspartate aminotransferase (AST) 137 U/L, N 1034 U/L; alanine aminotransferase (ALT) 78 U/L, N 535 U/L; alkaline leukocyte phosphatase (ALP) 267 U/L, N 20140 U/L, and gammaglutamyltranspeptidase (GT) 505 U/L, N 051 U/L]. Further exams at this period revealed colpocephaly [cranial computed tomography (CT)], infantile hypertrophic pyloric stenosis [abdominal ultrasound (US)] and interatrial communication (ECO). At one-and-a-half months of age, pyloromyotomy was performed, but vomiting episodes persisted. At this time, she still presented with lactic acidemia (5.0 mol/ml), metabolic acidosis, and mild hyperammonemia. Hemotransfusion was performed due to anemia. At 2 months and 4 days, hemoculture was positive for Staphylococcus epidermidis, and she was treated with antibiotics for 14 days. There was a small weight gain, but hepatic enzymes remained altered. At 3 months and 5 days, the girl was discharged but continued to gain weight slowly, although being fed with breast milk and supplementary

J Inherit Metab Dis (2010) 33:411419 Table 1 Urinary and plasma amino acid analysis by ion-exchange chromatography of the patient here reported Amino acid Urine 1 (nmol/mg creatinine) Urine 2 (nmol/mg creatinine) Reference valuesa (0 - 1m) (nmol/mg creatinine) 1241118 2482898 0146 104875 381373 81923 47200 28402 1331490 15411 2181795 3652267 Plasma 1 (nmol/ml) 313 263 181 70 99 430 nd 66 257 77 107 130

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Reference valuesa (0-1m) (nmol/ml) 90329 99395 1045 1060 29132 376709 1798 48160 55147 48211 92325 30138

Threonine Serine Citrulline Methionine Asparagine Glutamine Cystine Leucine Tyrosine Ornithine Lysine Histidine nd not detected
a

10600 7350 5494 nd 4470 5120 1359 539 3690 1238 3652 6427

2262 3416 638 nd 207 1540 1564 177.5 351.5 925.7 2683 2467

Metabolic Laboratory, Childrens Medical Center, Dallas, Texas, USA

formula. Vomiting episodes twice a day and occasional diarrhea persisted. At 4 months and 9 days, she was readmitted owing to vomiting with traces of blood, hyperpyrexia, intense crying and pallor, dyspnea, and

dehydration. Besides hepatosplenomegaly, she presented with severe metabolic acidosis (pH 6.45; pCO2 32 mmHg; HCO3 2 mEq/L; BE 32 mEq/L) and bradycardia, which evolved to cardiac arrest and death. It was not possible to

Table 2 Clinical features found in 36 patients with fumaric aciduria described in the literature and in the case presented here Clinical findings Global developmental delay Cerebral abnormalities Hypotonia Dysmorphism Convulsive disorders a Microcephaly Feeding difficulties Failure to thrive Do not follow or fix Hematologic alterations b Macrocephaly c Vomiting Lethargy Gastrointestinal abnormalities Gestational history Prematurity (3336 weeks) Polyhydramnios Others e Patients 34/36 33/36 26/36 16/36 16/36 13/36 12/36 11/36 10/36 9/36 9/36 8/36 4/36 4/36 10/36 5/36 6/36 % 94 92 72 44 44 36 33 31 28 25 25 22 11 Present Case + + + + + +

References 120 119 2, 410, 12, 1419 3, 9, 1113, 16, 18, 19 4, 79, 13, 18, 19, 20 2, 46, 8, 9, 1517, 19 2, 3, 610, 12, 14, 17 2, 3, 57, 9, 12, 1416 2, 8, 11, 14, 16 6, 11, 12, 16 11, 18 1 4, 6, 7, 10, 14 2, 7, 14 2, 11, 16 1, 4, 6, 7, 9, 13,15, 18 3, 6, 13, 17 6, 7, 9, 16, 19

+ + +f +

28 14 17

1: Whelan et al 1983; 2: Zinn et al 1986; 3: Walker et al 1989; 4: Gellera et al 1990; 5: Elpeleg et al 1992; 6: Bourgeron et al 1994; 7: Narayanan et al 1996; 8: Bonioli et al 1998; 9: Coughlin et al 1998; 10: Manning et al 2000; 11: Kerrigan et al 2000; 12: Zeman et al 2000; 13: Kimonis et al 2000; 14: Remes et al 1992 and 2004; 15: Loeffen et al 2005; 16: Phillips et al 2006; 17: Deschauer et al 2006; 18: Zeng et al 2006; 19: Maradin et al 2006; 20: De Meirleir et al 2006
All types of convulsions included and two patients were not responsive to drug treatment, b Neonatal polycythemia in five patients from the same family, Only two families; d malrotation of the bowel and esophageal dysmotility; e reduced fetal movements, excessive fetal weight, bleeding, intrauterine growth retardation, and leakage of amniotic fluid, f infantile hypertrophic pyloric stenosis
c

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Table 3 Cerebral abnormalities found in 33 patients with reported neuroimaging and in the case presented here Cerebral abnormalities Ventriculomegaly/ Hydrocephaly Cerebral Atrophies Open Sylvian Operculum a Polymicrogyria a Small brain Stem b Agenesis/ Hypoplasia of corpus callosum Brain cysts c Hypomyelination Others
d

Patients 27/33 24/33 10/33 9/33 8/33 6/33 6/33 5/33 2/33

Percent 82 73 30 27 24 18 18 15 6

This case + + -

References 19, 1116, 18, 19 25, 7, 911, 13, 14, 1619 11, 16 11, 18 11, 16 3, 7,9, 16, 19 3, 11, 15 4, 7, 8, 16 9, 12

Authors references according Table 2. a : 8 from the same family; b : 6 from the same family; c : 3 choroid plexus cysts; 1 arachnoid cyst in the temporal fossa; 1 periventricular cysts; 1 cyst in occipital horn. d : Dandy-Walker malformation and lissencephaly

measure enzymatic activity and perform molecular analysis, since no laboratory in Brazil executed these exams. Postmortem exam was not allowed by the parents.

Clinical findings The literature group consisted of 40 patients from 27 families. Clinical and biochemical data were described in 36 patients, enzymatic activity determined in 31, and mutational data in 26. Of these 36 patients, 20 were females and 16 males, and their main clinical features and cerebral abnormalities are shown in Tables 2 and 3, respectively. Cerebral abnormalities were observed in 33 patients and the three remaining were mentally impaired (Whelan et al 1983; Bourgeron et al 1994; De Meirleir et al 2006). These malformations in fumaric aciduria could be a result of

Overview: clinical, laboratorial, and mutational features in fumarase-deficient patients Data from the literature were compiled, compared with the case here reported, and discussed. It is important to stress that not all patients had complete description of clinical, biochemical, and mutational data.

Table 4 Dysmorphic features described in 15 of 16 patients and in the case presented here

Dysmorphic Features Depressed nasal bridge Frontal bossing Hypertelorism Notched/anteverted naresa Low-set/anteverted/prominent ears High-arched palatea Bitemporal narrowing Overridging sutures Metopic ridgingb Triangular lower fasciesb Brachycephaly b Dolichocephaly Micrognathia Hypotelorism Pointed chin Camptodactyly Polydactyly Colpocephaly Hooked nose

Patients 12/15 10/15 9/15 5/15 4/15 3/15 3/15 2/15 2/15 2/15 2/15 1/15 1/15 1/15 1/15 1/15

Percent 80 67 60 33 27 20 20 13 13 13 13 7 7 7 7 7

Percent total 33 28 25 14 11 8 8 6 6 6 6 3 3 3 3 3

This case + + + + + + +

References 11, 11, 11, 11 12, 11 16, 18, 19 12, 18 19 13, 18, 19

References according Table 2.


a

Described in one family with eight affected members (Kerrigan et al 2000), b monozygotic twins

13, 16 16 16 16 16 13 12 13 13 10

J Inherit Metab Dis (2010) 33:411419 Table 5 Altered substances in physiological fluids reported in patients with fumaric aciduria Altered organic acid Number of patients Urine Fumaric Succinic 2-ketoglutaric Adipic Citric Latic Malic Pyruvic Suberic 3-OH-butyric 4-OH-phenylacetic Acetoacetic Methylmalonic References according to Table 2 CSF cerebral spinal fluid
a

415

This case Plasmaa 5 1 4 3 2 1 CSFa 1 5 2 +

References

36/36 6/36 6/36 2/36 2/36 3/36 3/36 2/36 2/36 1/36 1/36 1/36 1/36

120 24, 7, 9, 12, 13 3, 4, 6, 13, 15 2, 4 2, 7 24, 6, 7, 8, 12, 17 4, 8, 13 4, 68, 15 2, 4 7 7 6, 7 15

+b

Organic acid analyses were not performed in all patients b plasma

aerobic energy loss during early embryogenesis (Phillips et al 2006). Global developmental delay was present in almost all patients reviewed in the literature. From the two remaining patients, one had only seizures and the other one died at 17 h of life (Coughlin et al 1998; Manning et al 2000). Speech impairment was observed in 14 patients (from six different families), some being unable to speak, some being dysarthric, and others restricted to few words
Fig. 1 Metabolic pathways involving fumarate
SAICAR
ASL

(Whelan et al 1983; Maradin et al 2006; Coughlin et al 1998; Kerrigan et al 2000; De Meirleir et al 2006; Zeng et al 2006). Abnormal posture was seen in seven hypotonic patients, five showing opisthotonus. Two patients presented with areflexia associated with hypotonia. (Narayanan et al 1996; Manning et al 2000). Other neurologic findings were bilateral Babinski sign, spasticity, choreoathetoid moveAICAR FAICAR IMP

CYTOSOL
ASPARTATE + GTP

GDP + Pi

MALEYLACETOACETATE

MAI

FUMARYL ACETOACETATE

AMP

ASL

ADENYLOSUCCINATE

HGO

O2

FAH

HOMOGENTISATE
CO2
4HPPD

ACETOACETATE

S-MALATE
ME

FH

SCS

FUMARATE
SDH

SUCCINATE

SUCCINYL-CoA
CO2
2-CGDH

MITOCHONDRION

O2

pOHPHENYLPYRUVATE
TAT

PYRUVATE + CO2

FUMARATE
FH
AS

-KETOGLUTARATE

TYROSINE

ARGININO SUCCINATE

ARGININE

KREBS CYCLE
S-MALATE
ME MDH

CO2
ICDH

GLUCOSE LACTATE

ISOCITRATE
AC

CITRATE

PYRUVATE
PDHC

ASS

UREA CYCLE
CITRULLINE
OT

ATP + HCO- 3
PC

H2O UREA

CS

ACETIL-CoA

ADP + Pi

ASPARTATE ORNITHINE

OXALOACETATE PYRUVATE + CO2 -KETOGLUTARATE


AST GD

AMINO ACID
AMINOTRANSFERASE

GLUTAMATE NH 3 CARBAMOYL PHOSPHATE


CPSI

-KETO ACID

HCO- 3 + ATP

ADP

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ments, ataxia, and dystonic tetraplegia (Walker et al 1989; Narayanan et al 1996; Kerrigan et al 2000; Manning et al 2000; Remes et al 2004; Loeffen et al 2005; Maradin et al 2006). Of 11 patients with reported EEG, ten showed epileptiform features (Zinn et al 1986; Gellera et al 1990; Elpeleg et al 1992; Remes et al 1992; Bonioli et al 1998; Kimonis et al. 2000; Loeffen et al 2005; Deschauer et al 2006; Phillips et al 2006). Sixteen patients from eight different families had some degree of dysmorphia, and craniofacial dysmorphic features were present in all of them (Table 4). It must be observed that in disorders with abnormalities in cell energy production, dysmorphic features may occur. However, the underlying mechanism in fumaric aciduria is unknown (Kerrigan et al 2000; Peters and Kahler 2003). Prematurity was a relevant gestational feature, have being associated with polyhydramnios in two cases. In general, the main fetal anomalies associated with polyhy-

dramnios are a consequence of disorders that affect gastrointestinal, musculoskeletal and the central nervous system (CNS) (Kyle et al. 1997). Angulation of the frontal horns and optic nerve hypoplasia/pallor were described in a consanguineous family that had eight affected members; these features are unique in study group (Kerrigan et al 2000). By the time of their respective reports, ten patients had died early (28%), seven of them being younger than 1 year, such as the case here reported. The cause of death was described only in seven patients and in general was due to infections (3), cardiac arrest (3), or prolonged apnea (1). Laboratorial findings Metabolite alterations in physiological fluids reported in the literature in patients with fumaric aciduria are described in Table 5.

Table 6 Mutations found in 25 patients with fumarase deficiency (adapted from Bayley et al 2008) Original reference Original mutation description DNA 1, 3, 8, 9, 11 5, 7, 10 6, 9, 12 10 2 8, 9 3 3, 9 6 9 3 3 3 11 13 9 1 13 3 14 14 1302insAAA,1433insAAA, AAAins435 998A>C, 1127A>C 392C>G, 492C>G 1078C>T 955G>C 698G>A 1148A>T 560A>G 779T>C 2-slice exon 2 66del74 793G>A 806T>G 824A>T 976C>T 1029_1031delAGT 1373G>A 1078C>T Intronic mutation c.1391-269A>G Protein 434insLys, 435insLys, 477insLys Gln13Pro, Gln333Pro, Gln376Pro Pro131Arg His360Cys Glu319Gln Arg190His, Arg233His Asp383Val Lys187Arg Leu260Ser Ala265Thr Phe269Cys His275Leu Pro326Ser Arg58X Arg190Cys Gln343His/ 344delVal Trp458X c.1431_1433dup c.1127A>C c.521C>G c.1207C>T c.1084G>C c.698G>A c.1274A>T c.689A>G Exon 2 splice variant c.195_268del c.922G>A c.935T>G c.953A>T c.1105C>T c.301C>T c.697C>T c.1158_1060delAGT c.1499G>A c.1204C>T p.Lys477dup p.Gln376Pro p.Pro174Arg p.His402Cys p.Glu362Gln p.Arg233His p.Asp425Val p.Lys230Arg p.Leu303Ser p.Lys66fs p.Ala308Thr p.Phe312Cys p.His318Leu p.Pro369Ser p.Arg101X p.Arg233Cys p.Gln386_ Val387delinsHis p.Trp500X p.His402Tyr 7/26 5/26 3/26 2/26 2/26 2/26 2/26 2/26 1/26 1/26 1/26 1/26 1/26 1/26 1/26 1/26 1/26 1/26 1/26 1/26 1/26
a,b

DNA change*

Protein mutation*

Number of patients

e a c

1: Gellera et al 1994; 2: Bourgeron et al 1994; 3: Coughlin et al 1998; 4: Manning et al 2000; 5: Zeman et al 2000; 6: Kimonis et al 2000; 7: Remes et al 2004; 8: Loeffen et al 2005; 9: Pollard et al 2005; 10: Phillips et al 2006; 11: Deschauer et al 2006; 12: Zeng et al 2006; 13: Maradin et al 2006; 14: De Meirleir et al 2006 * Some of the reported FH mutations were traditionally based on the mature protein rather than the primary translation product, which also contains the signal peptide of 43 amino acids, for mitochondrial signaling. They were corrected for the mitochondrial isoform, considered the canonic form,a monozygotic twins with compound heterozygous mutation; b,c,d siblings; e one case of paternal partial isodisomy

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The massive excretion of fumaric acid in urine is common to all patients (15- to 1,000-fold higher when compared with normal range). In one patient, fumaric acid elevation was not observed with the first symptoms but only appeared later (Narayanan et al 1996). Some considerations regarding other metabolite changes in patients with fumaric aciduria are presented below. Other organic acids were also shown to be altered in fumaric aciduria, with urinary ketoglutaric and succinic acids being the most relevant besides fumaric acid (Table 5). These findings were probably due to secondary enzymatic inhibition of succinate dehydrogenase (SDH). This enzyme belongs to complex II and is part of the respiratory chain and Krebs cycle; it catalyzes the conversion of succinic acid to fumaric acid (Fig. 1). The reverse reaction is barely detectable in human cells and tissues in normal conditions (Brire et al 2005). An unexpected finding from the literature was the presence of malic acid in three patients. This could be explained by the accumulation of acetyl-coenzyme A (CoA) in mitochondria that inhibits pyruvate dehydrogenase complex (PDHC) and stimulates pyruvate carboxylase (PC). The result is increased production of oxaloacetic acid, which is converted to malic acid by malate dehydrogenase (MDH) (Gellera et al 1990). Another alternative pathway would be the mitochondrial and cytosolic malic enzyme converting pyruvic acid to malic acid. This bidirectional enzyme is highly stimulated when the mitochondrial levels of fumaric and succinic acids are increased (Taroni et al 1988) (Fig. 1). Accumulation of fumaric acid due to FH deficiency may affect several other metabolic pathways. The purine nucleotide cycle, necessary for cellular replication, aids in tight regulation of cellular metabolism by controlling the amounts of available citric acid cycle intermediates and the
Fig. 2 Mutations observed on fumarate hydratase enzyme (mitochondrial isoform)

amount of free adenosine monophosphate (AMP) (Toth and Yeates 2000; Tsai et al 2007). In purine de novo biosynthesis, adenylosuccinate lyase (ASL, E.C. 4.3.2.2) catalyzes two similar reactions producing 5-aminoimidazole-4-carboxamide ribotide (AICAR), AMP, and fumaric acid (Fig. 1). ASL could be affected by the increased amount of fumaric acid, which is a weak and noncompetitive inhibitor of this enzyme (Barnes and Bishop 1975). In one patient, two succinylpurine derivatives, 5-aminoimidazole-(N-succinylcarboxiamide) ribotide (SAICAR) and adenylosuccinate, were found in cerebral spinal fluid (CSF). In urine of two patients, these derivatives were not detected, as such metabolites do not cross the blood-brain barrier (Elpeleg et al 1992, Zeman et al 2000). Zeman and coworkers considered that the accumulation of these succinylpurine derivatives could have a profound effect on impairment of the CNS in fumaric aciduria (Zeman et al 2000). SAICAR, which is a substrate for ASL, was considered a potential neurotoxic agent to pyramidal neurons of rat hippocampus (Stone et al 1997). From 12 literature cases in which amino acid analysis was performed, three patients, two being siblings, showed altered results. Bourgeron and coworkers reported that both siblings had slightly elevated phosphoethanolamine, threonine, alanine, valine, serine, and glycine in blood and urine. Aspartate and asparagine were low in these two patients blood and elevated in urine of one of them (Bourgeron et al 1994). In the third patient, only urinary amino acids were altered, but these were not discriminated (Narayanan et al 1996). The patient here reported showed an increased urinary excretion of several amino acids (Table 1). Another interesting finding was the elevated levels of tyrosine, methionine, and citrulline in plasma not

10 20 30 40 50 60 MYRALRLLAR SRPLVRAPAA ALASAPGLGG AAVPSFWPPN AARMASQNSF RIEYDTFGEL 70 80 90 100 110 120 KVPNDKYYGA QTVRSTMNFK IGGVTERMPT PVIKAFGILK RAAAEVNQDY GLDPKIANAI 130 140 150 160 170 180 MKAADEVAEG KLNDHFPLVV WQTGSGTQTN MNVNEVISNR AIEMLGGELG SKIPVHPNDH 190 200 210 220 230 240 VNKSQSSNDT FPTAMHIAAA IEVHEVLLPG LQKLHDALDA KSKEFAQIIK IGRTHTQDAV 250 260 270 280 290 300 PLTLGQEFSG YVQQVKYAMT RIKAAMPRIY ELAAGGTAVG TGLNTRIGFA EKVAAKVAAL 310 320 330 340 350 360 TGLPFVTAPN KFEALAAHDA LVELSGAMNT TACSLMKIAN DIRFLGSGPR SGLGELILPE 370 380 390 400 410 420 NEPGSSIMPG KVNPTQCEAM TMVAAQVMGN HVAVTVGGSN GHFELNVFKP MMIKNVLHSA 430 440 450 460 470 480 RLLGDASVSF TENCVVGIQA NTERINKLMN ESLMLVTALN PHIGYDKAAK IAKTAHKNGS 490 500 510 TLKETAIELG YLTAEQFDEW VKPKDMLGPK

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previously reported in the literature. Elevated tyrosine and methionine levels could be due to the secondary inhibition of the last enzyme of the tyrosine catabolic pathway, fumarylacetoacetate hydrolase (FAH, E.C.3.7.1.2.), by increased levels of fumaric acid (Fig. 1). This inhibition could also result in elevation of several hepato- and neurotoxic metabolites, such as observed in tyrosinemia type I. The main features of this disease, renal Fanconi syndrome, and hepatic problems (hepatomegaly, jaundice, and cholestasis) (Mitchell et al 2001), were also presented by the patient here reported. Hepatomegaly was described in two literature patients, and one of them also presented with jaundice and cholestasis (Walker et al 1989; Zeman et al 2000). Another consequence of fumaric acid accumulation could be a diminished activity of argininosuccinase (AS, E.C. 4.3.2.1), which would explain the significant plasma citrulline elevation (Fig. 1). Some symptoms, such as hyperammonemia and vomiting, common to urea cycle defects, were presented by the patient. Hyperammonemia was reported only in two patients from the literature (Zinn et al 1986, Walker et al 1989). Severe metabolic acidosis was present in the patient described here from birth and in only two literature patients (Gellera et al. 1990, Narayanan et al 1996). The measured residual FH activity in the literature patients (n=31) ranged from not detectable to 36% of control mean (average 5%). Heterozygous parents had the activity ranging from 30% to 74% of the control mean. However, it has been observed that fumaric acid excretion is not correlated with residual enzyme activity or severity of the clinical picture (Bonioli et al 1998). Mutational findings Human FH gene consists of ten exons encoding 510 amino acids. The first exon (exon 0) encodes a mitochondrial localization signal peptide of 43 amino acids (UniProt, 2009). The mutations related to FH deficiency reported in the literature so far are shown in Table 6 (modified from Bayley et al 2008). It can be observed that FH genotype is very heterogeneous, and most mutations are concentrated at the C-terminus of the FH enzyme (Fig. 2). Twenty-one different mutations related with congenital fumarase deficiency were described. In two patients, only one allele mutation was found at the time of their reports (Coughlin et al 1998). Fourteen mutations were missense. The remaining mutations were nonsense, duplication, frameshift, splice site, and deletion/insertion. Twelve patients were homozygous for the mutations Gln376Pro, Pro174Arg, Glu362Gln, Asp425Val, and Lys230Arg. Mutation p.Lys477dup was the most frequent, being observed in seven heterozygous nonrelated patients (27%).

Final considerations It must be considered that some of the unusual findings of the patient here described could also be due to another genetic defect and not exclusively to fumaric aciduria, since the parents are consanguineous. Fumaric aciduria has a broad spectrum of clinical manifestations, although the great majority of patients present with global developmental delay and cerebral abnormalities. Biochemically, however, elevated excretion of fumaric acid has been found in all patients, being therefore considered a hallmark of this disorder. The significant accumulation of fumaric acid due to FH deficiency may affect several metabolic pathways, including de novo purine nucleotide biosynthesis, tyrosine catabolic pathway, urea cycle, and citric acid cycle. Some possible explanations, regarding clinical features and abnormal metabolite levels in fumaric aciduria patients, are presented. An interesting finding observed in the patient here reported was the alteration of several plasma and urinary amino acid levels. Such changes allowed a few pertinent considerations that may expand the current knowledge of this rare disease. Although organic acid analysis is the first and main step to be carried out in patients suspected of having fumaric aciduria, it is suggested that amino acid analysis in body fluids be also performed. Moreover, it would be interesting to consider the hepatic picture owing to the signs described in the case presented here.
Acknowledgments We thank Francisco Radler de Aquino Neto, Luis Nelson L F Gomes, DLE (Diagnsticos Laboratoriais Especializados), Markta Tesaov and Jean-Pierre Bayley for their contribution to the study.

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