Glutaric aciduria type 1: Clinical, biochemical and molecular findings in patients from Israel

ARTICLE IN PRESS E U R O P E A N J O U R N A L O F PA E D I AT R I C N E U R O L O G Y

11 (2007) 81 – 89

Official Journal of the European Paediatric Neurology Society

Original article

Glutaric aciduria type 1: Clinical, biochemical and molecular findings in patients from Israel Stanley H. Kormana,b,, Cornelis Jakobse, Patricia S. Darmine, Alisa Gutmana, Marjo S. van der Knaapf, Ziva Ben-Neriahc, Imad Dweikatg, Isaiah D. Wexlerd, Gajja S. Salomonse a

Department of Clinical Biochemistry, Hadassah—Hebrew University Medical Center, Jerusalem, Israel Metabolic Diseases Unit, Hadassah—Hebrew University Medical Center, Jerusalem, Israel c Department of Human Genetics, Hadassah—Hebrew University Medical Center, Jerusalem, Israel d Department of Pediatrics Mount Scopus, Hadassah—Hebrew University Medical Center, Jerusalem, Israel e Department of Clinical Chemistry ,VU University Medical Center, Amsterdam, The Netherlands f Department of Child Neurology, VU University Medical Center, Amsterdam, The Netherlands g Department of Pediatrics, Makassed Islamic Hospital, Jerusalem, Israel b

art i cle info

ab st rac t

Article history:

Glutaric aciduria type 1 (GA1) is a rare cerebral organic aciduria which typically manifests

Received 2 November 2006

as an acute encephalopathic crisis followed by profound long-term neurological handicap.

Accepted 18 November 2006

We report the diagnosis of 12 new patients from a single laboratory in Israel during a 5-year period. Eleven of the 12 were of Palestinian origin, and only two were related. One patient

Keywords:

was asymptomatic whilst one was mildly, one moderately and nine severely affected, two

GCDH mutations

of whom had unusual MRI findings. Two patients had normal glutaric acid excretion and

Prenatal diagnosis

normal blood glutarylcarnitine levels yet glutarylcarnitine excretion was increased,

Acylcarnitines

indicating its utility as a diagnostic marker. Four novel GCDH mutations (Thr193_Arg194in-

Glutarylcarnitine

sHis, Asn329Ser, Thr341Pro, Met405Val) and five previously reported mutations (Ser119Leu,

Glutaric acid

Leu283Pro, Ala293Thr, Gly390Arg and Thr416Ile) were identified. Severely and mildly

3-hydroxyglutaric acid

affected or even asymptomatic patients shared the same genotypes (Thr416Ile/Thre416Ile and Aal293Thr/Thr193_Arg194insHis). Knowledge of the responsible mutation enabled successful prenatal diagnosis on chorionic villous DNA in three families. In conclusion, GA1 is genetically heterogeneous and has a relatively high incidence in the Palestinian population, reflecting the historical tradition of marriages within extended kindreds, particularly in isolated villages. Additional genetic and/or environmental factors must account for the phenotypic heterogeneity in patients with the same genotype. The diagnosis was not suspected in the majority of cases despite typical clinical and/or neuroimaging features, suggesting that glutaric aciduria may be under-diagnosed. Greater awareness of glutaric aciduria amongst pediatricians, neonatologists and radiologists is the key to identifying the disorder in the presymptomatic phase and preventing its catastrophic consequences. & 2006 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.

Corresponding author. Department of Pediatrics Mount Scopus, Metabolic Diseases Unit, Hadassah—Hebrew University Medical Center, POB 12000, Jerusalem 91120, Israel. Tel.: +972 2 6778420; fax: +972 2 6779018. E-mail address: [email protected] (S.H. Korman). 1090-3798/$ - see front matter & 2006 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ejpn.2006.11.006

ARTICLE IN PRESS 82

1.

E U R O P E A N J O U R N A L O F PA E D I AT R I C N E U R O L O G Y

Introduction

The neurometabolic disorder glutaric aciduria type 1 (GA1, OMIM ]231670) is an autosomal recessively inherited defect in the pathway of lysine, hydroxylysine and tryptophan catabolism caused by deficiency of glutaryl-CoA dehydrogenase (EC 1.3.99.7) encoded by the GCDH gene. It is one of the cerebral organic acidurias, a distinctive subgroup of organic acidurias which manifests predominantly as an encephalopathy rather than an acute disturbance in systemic metabolic homeostasis. The clinical phenotype of GA1 has been summarized in a number of patient cohort descriptions.1–4 It typically presents in a developmentally normal infant towards the first birthday as an acute encephalopathic crisis following an intercurrent illness, with permanent residual sequelae including dystoniadyskinesia, seizures and developmental regression or delay. The biochemical diagnosis of GA1 is based on detection of large quantities of glutaric acid (GA) and 3-hydroxyglutaric acid (3-HGA) excreted in the urine and elevated levels of glutarylcarnitine in the blood. Some patients, however, have normal GA excretion and only slightly elevated 3-HGA excretion5 and may also have normal glutarylcarnitine levels.6 A number of hypotheses have been proposed to explain the pathogenesis of the disorder and the stroke-like acute striatal necrosis that underlies the encephalopathic crisis. These include NMDA receptor-mediated excitotoxicity induced by 3-HGA7 or by the kynurenine pathway product quinolinic acid,8 glutaconylCoA-mediated neurotoxicity9 and disruption of vascular endothelial integrity by 3-HGA.10 Recently, striatal injury was induced in a GCDH knockout mouse model by administration of a high-protein or high-lysine diet and was attributed to extremely high levels of GA accumulating in the brain,11 a finding also observed in human post-mortem studies.12 Once the encephalopathic crisis has occurred, management of GA1 patient is limited to conservative measures as the striatal damage is irreversible. If the diagnosis can be made in the presymptomatic phase, appropriate management can prevent the catastrophic neurological deterioration in the majority of cases.1,4,13 This prophylactic management involves administration of carnitine supplements, restriction of dietary lysine and/or protein intake and adherence to an emergency regimen protocol designed to reverse the catabolic state during acute febrile or vomiting episodes by administering intravenous fluids with high glucose concentration as well as lipids and carnitine. We present the clinical features and the biochemical and neuroimaging findings of a series of GA1 patients from Israel and report recurrent and novel GCDH mutations identified in these patients. Our findings indicate that GA1 is relatively common in the Palestinian population but that the diagnosis is often delayed or missed due to lack of familiarity with this disorder amongst pediatricians and allied professionals.

2.

Methods

2.1.

Patients

This study comprises all GA1 patients diagnosed by the metabolic laboratory at Hadassah—Hebrew University Medi-

11 (2007) 81 – 89

cal Center during a 5-year period ending in mid-2004. Patient ]1 has been previously reported.14,15

2.2.

Biochemical analyses

Qualitative analysis of urine organic acids was performed by gas chromatography – mass spectrometry (GC-MS) following a standard ethyl acetate/diethyl ether extraction and trimethylsilyl derivatization. GA concentration in urine was determined by GC-MS using a stable isotope dilution method. HGA in urine was quantified by a stable-isotope dilution method based upon negative chemical ionization GC-MS and selected ion monitoring for 3-HGA as its pentafluorobenzyl (PFB) ester.16 Blood spot acylcarnitine concentrations, including glutarylcarnitine (C5-dicarboxylic carnitine) were determined by electrospray ionization tandem mass spectrometry (ESIMS-MS). Urine samples were also subjected to ESI-MS-MS analysis by multiple reaction monitoring for selected metabolites using positive-ion analysis for detection of amino acids and acylcarnitines (including glutarylcarnitine) in dried and butylated samples, and negative-ion analysis for detection of organic acids (including glutaric and HGA) in samples analyzed directly after dilution with the mobile phase (50% acetonitrile).17

2.3.

GCDH gene mutation analysis

Mutational analysis of the GCDH gene was performed on genomic DNA isolated from peripheral blood. The 11 exons and flanking intronic regions of the GCDH gene were amplified, purified and sequenced using the primer sequences and PCR conditions as previously described.14 The obtained sequences were aligned using the mutation explorer software (SoftGenetics Inc., PA, USA) and the genomic GCDH sequences as references (http://www.ncbi.nlm.nih.gov/GenBank: GI 3150003, GI 2316108, GI 2316109, GI 2316110).

3.

Results

The clinical, biochemical and molecular findings in the patients are summarized in Table 1.

3.1.

Clinical and demographic findings

Twelve patients were diagnosed from eleven families. Their ethnic origin was Palestinian Arab in eleven and Jewish in one (patient ]12). Nine patients were male and three (]7, ]10 and ]11) were female. Nine of the patients had the typical course of symptomatic GA1. Three of them (]2, ]11 and ]12) were diagnosed during or shortly after their acute encephalopathic crisis as a result of general metabolic screening performed for investigation of an acute encephalopathy of unknown origin. Six of the patients (]1, ]4–7 and ]10) were diagnosed when evaluated for chronic or progressive neurological symptoms including dystonia, seizures, spasticity and developmental delay; in at least three of these (]1, ]6 and ]10), it was possible to retrospectively elicit a history of a preceding acute encephalopathic crisis for which no etiology had been established.

Table 1 – Clinical, biochemical and molecular findings in twleve glutaric aciduria type 1 patients Age at diagnosis

]

4m 28 m 14 m 10 m 3 yr

7 8

1 yr 7d

9

30 yr

10

3 yr

11 12

8m 5m

Genetic counseling (uncle of patient ]8); borderline IQ Severe dev. delay post-acute infantile encephalopathy Acute encephalopathy Convulsions with fever, dystonia

Urine GA (mmol/ mol creatinine)

Urine 3-HGA (mmol/ mol creatinine)

Plasma free/total Carnitine (mmol/L)

Blood Glutarylcarnitine (mmol/L)

GCDH genotype

Typical

Severe

6–50y

33

15.7/32.7

o0.02

L283P/L283P

Typical Typical Typical Typical Diffuse white matter Typical Typical

Severe Normal Severe Severe Severe

z

z

6811 1199

160 61

z

z

13344

197

5.9/11.4 2.3/5.5 5.8/10.3 3.9/7.4 3.0/—

0.41 0.57 0.54 0.35 0.58

G390R/G390R T416I/T416I T416I/T416I T416I/T416I T416I/T416I

Died Moderate

z

z

243

44

4.7/8.8 6.6/14.6

nd 0.98

Mild

728

49

4.7/7.9

1.01

Severe

1830

70

9.8/12.0

0.76

T4161/A293T A293T/ T193_R194insH A293T/ T193_R194insH T341P/T341P

Severe Died

z

z

6

15

4.0/13.1 18.1/30.5

0.57 0.11

N392S/N392S S119L/M405V

Subependymal nodules Typical Typical Basal ganglia only

 Normal ranges of metabolite concentrations: urine GA (glutaric acid) 0.5–11 mmol/mol creatinine; urine 3-HGA (3-hydroxyglutaric acid) 0.9–4.5 mmol/mol creatinine; plasma-free carnitine

25–35 mmol/L, total carnitine 35–45 mmol/L; blood spot glutarylcarnitine o0.07 mmol/L (carnitine and glutarylcarnitine results are basal levels prior to oral carnitine supplementation). [GA] elevated only on valproate therapy. z Markedly increased excretion on qualitative urine organic acid analysis, but not quantified; nd, not done. y

ARTICLE IN PRESS

2 3 4 5 6

Severe dev. delay and seizures post-acute infantile encephalopathy Acute encephalopathy, seizures Macrocephaly; normal development Macrocephaly, dev. delay Macrocephaly, dystonia Severe dev. delay post-acute encephalopathy at age 4 m Severe dev. delay, dystonia Macrocephaly at birth

Outcome

1 1 (20 07) 81 – 8 9

14 m

Neuroimaging

E U R O P E A N J O U R N A L O F PA E D I AT R I C N E U R O L O G Y

1

Presentation

83

ARTICLE IN PRESS 84

E U R O P E A N J O U R N A L O F PA E D I AT R I C N E U R O L O G Y

Three of the patients (]3, ]8 and ]9) had an atypical presentation. Patient ]8 was investigated in the first week of life because of macrocephaly and a history that two of the mother’s previous pregnancies had been terminated due to ultrasound findings of anencephaly and multiple congenital anomalies (in one of these GA1 was excluded post-factum by testing DNA from the abortus for the GCDH mutations identified in patient ]8). This baby (]8) subsequently had seizures, hypotonia and developmental delay. The remaining two patients (]3 and ]9, see below) were essentially asymptomatic. In only three patients (]3, ]5 and ]12) was the specific diagnosis of GA1 ever considered by the medical team referring samples to the laboratory for general metabolic screening. Patient ]3 was found on routine examination to have macrocephaly and underwent brain CT examinations at age 13 and 16 months which revealed bilateral fronto-temporal atrophy with arachnoid cysts and subdural fluid collections. The diagnosis of GA1 was never considered and he underwent bilateral craniotomy with drainage of bloodstained fluid from both subdural spaces. Only 12 months later was the diagnosis of GA1 first suspected when he was evaluated by a pediatric neurologist. Apart from the finding of macrocephaly, neurological examination and developmental assessment were entirely within normal limits. He was started on treatment with mild restriction of dietary protein intake and oral carnitine supplementation. His subsequent progress has been uneventful, and at age 9 yr he is an average student attending a normal school. Patient ]9 presented together with his wife, who is his first cousin and was in the seventh week of her first pregnancy. They sought genetic counseling regarding the need for prenatal diagnosis of GA1, as he is the maternal uncle and she the paternal aunt of GA1 patient ]8. As patient ]8 had already been shown to be compound heterozygous for two GCDH mutations, they were counseled to undergo carrier testing for these mutations in order to determine whether they were at risk of having an affected child. This revealed that the mother was a carrier of one of the mutations but that the father was compound heterozygous for both mutations. Metabolite testing (Table 1) confirmed that he does indeed have GA1. Physical and neurological examination were generally normal; his IQ was borderline. His brain MRI findings are discussed in Section 3.3.

3.2.

Biochemical findings

The results of metabolite determinations are presented in Table 1. Most patients had moderate to massive GA excretion and markedly increased 3-OH-GA excretion. Two patients (]1 and ]12), however, had normal (o11 mmol/mol creatinine) GA excretion; one of them (]1) had mildly elevated GA excretion (50 mmol/mol creatinine) whilst receiving valproic acid treatment for seizures but GA excretion was within normal limits when off treatment (valproic acid treatment is known to cause elevation of urinary GA excretion).18 Both of these patients had elevation of 3-HGA excretion (15 and 33 mmol/mol creatinine, respectively, normal o4.5), but to a lesser extent than the other patients (range 44–197 mmol/mol creatinine).

11 (2007) 81 – 89

All patients had low plasma free carnitine levels and an abnormally elevated esterified to free carnitine ratio. The plasma free carnitine deficiency was severe (i.e., o10 mmol/L, normal value 25–35 mmol/L) in all except the two patients (]1 and ]12) in whom urine GA excretion was normal. Despite the severe free carnitine deficiency, the blood spot glutarylcarnitine (C5-dicarboxylic carnitine) level was markedly elevated (range 0.35–1.01 mmol/L, normal o0.07 mmol/L) except in the two patients with normal GA excretion, who had normal or only minimally elevated levels. In summary, when compared to the patients with marked glutaric aciduria, the two GA1 patients with normal GA excretion had only mildly abnormal excretion of 3-HGA, only minor carnitine deficiency and normal or only minimally elevated blood spot glutarylcarnitine. In addition to the above examinations, urine from six patients (]1, ]2, ]4, ]6, ]9 and ]12) was also examined by tandem mass spectrometry. On negative ion ESI-MS-MS, C5-dicarboxylic acid concentration was normal in patients ]1 and ]6, corresponding with the absence of glutaric aciduria on GC-MS examination of urine organic acids. All six had elevated hydroxyglutaric acid excretion. Positive ion ESI-MSMS analysis revealed an increased excretion of the C5-dicarboxylic acylcarnitine species, consistent with glutarylcarnitine, in all six patients, including the two with normal GA excretion and normal or virtually normal blood glutarylcarnitine.

3.3.

Neuroimaging

Most patients had typical neuroimaging features of GA119,20 including macrocephaly, brain atrophy, widely open pretemporal and Sylvian fissure CSF spaces, extracerebral fluid collections, subependymal cysts, basal ganglia lesions, delayed myelination and white matter signal abnormalities. Nevertheless, the diagnosis of GA1 was seldom considered and two patients (]3 and ]5) underwent unnecessary neurosurgical drainage, a potentially hazardous procedure in this disorder.21 Two severely affected patients had unusual MRI findings. An MRI study performed in patient ]6 at age 22 months, 18 months after his acute encephalopathic crisis, demonstrated typical Sylvian fissure and pre-temporal abnormalities and atrophic, virtually non-existent basal ganglia. In addition, the cerebral white matter was diffusely abnormal and decreased in volume and there were signal changes in the mid-brain and dorsal part of the pons. Patient ]12 had an MRI study at age 8 months during his acute presentation with fever, convulsions and dystonic movements. Prominent signal abnormalities were evident in the caudate nucleus, putamen and globus pallidus but the typical Sylvian fissure and pretemporal abnormalities were lacking. The MRI scan of patient ]9, diagnosed fortuitously at age 30 yr, revealed patchy signal changes in the corpus callosum with wart-like mass lesions extending from the ependymal lining into the lateral ventricles in the upper part of the ventricular system and showing some contrast enhancement (Fig. 1). The lesions were somewhat reminiscent of, although smaller than, the subependymal nodules seen in tuberous sclerosis; the patient did not exhibit any

ARTICLE IN PRESS E U R O P E A N J O U R N A L O F PA E D I AT R I C N E U R O L O G Y

cutaneous stigmata of tuberous sclerosis. The nature and significance of this finding, not previously reported in GA1, remains uncertain.

3.4.

GCDH mutation analysis

Mutations were detected at both alleles from all 12 patients (Table 1). In total, nine different mutations were identified (Table 2), four of which have not been previously reported. These consisted of an in-frame trinucleotide insertion (c. 578_579 insTCA) and three missense mutations (Thr341Pro, Asn392Ser and Met405Val) which each alter a GCDH residue conserved in primates, dog, rat, mouse, mosquito and other species. Furthermore, these missense mutations were not detected in 210 control alleles. Of the five previously reported mutations, four (Leu283Pro, Ala293Thr, Gly390Arg and Thr416Ile) had previously been identified in the Palestinian population.22 Thr416Ile was the commonest mutation detected, being present on 9 of the 22 alleles from the 11 families. The patients from four apparently unrelated families were Thr416Ile homozygous; one had normal outcome whereas the other three had severe neurological sequelae.

3.5.

85

1 1 (20 07) 81 – 8 9

Prenatal diagnosis

Prenatal diagnosis was performed in three families. Examination of DNA isolated from CVS tissue in a subsequent pregnancy of the mother of patient ]11 revealed that the fetus did not carry the p.Ans392Ser mutation and a normal infant was subsequently born. Analysis of CVS DNA in a subsequent pregnancy of the mother of patient ]2 revealed the presence of the homozygous p.Gly390Arg mutation and the pregnancy was terminated. Prenatal diagnosis performed in the first pregnancy of the wife of patient ]9 revealed that the fetus was a carrier, as expected, of the c.877G4A (p.Ala293Thr) mutation found in both parents, but did not carry the second mutation, c.578_579insTCA, found in the affected father. This predicted an unaffected fetus but unfortunately the mother miscarried 2 weeks after the CVS procedure.

4.

Discussion

In a period of 5 yr, our laboratory made a diagnosis of GA1 in 12 new patients, 11 of whom were of Palestinian descent. Considering that three additional laboratories in Israel perform diagnostic investigations for inborn errors of metabolism

Fig. 1 – MRI brain scan of patient ]9. The MRI scan of patient ]9, diagnosed fortuitously at age 30 yr, showing patchy signal changes in the corpus callosum with wart-like mass lesions (arrowheads) extending from the ependymal lining into the lateral ventricles.

Table 2 – GCDH gene mutations No. 1 2 3 4 5 6 7 8 9

Nucleotide change

Exon

c.356C4T c.578_579 insTCA c.848T4C c.877G4A c.1021A4C c.1168G4C c.1175A4G c.1213A4G c.1247C4T

V VI VII VIII IX X X X XI

 Not present in 210 control chromosomes.

Protein p.Ser119Leu p.Thr193_R194insHis p.Leu283Prp p.Ala293Thr p.Thr341Pro p.Gly390Arg p.Asn392Ser p.Met405Val p.Thr416Ile

Patient ] 12 8, 9 1/1 7, 8, 9 10/10 2/2 11/11 12 3/3, 4/4, 5/5, 6/6, 7

Reference 5

This report 22 22,30

This report 22

This report This report 22

ARTICLE IN PRESS 86

E U R O P E A N J O U R N A L O F PA E D I AT R I C N E U R O L O G Y

and that the awareness of GA1 and access to diagnostic facilities in the medical services in the Palestinian Territories is limited, we believe that the true number of patients in this population might be considerably higher. By comparison, only 28 patients in 27 yr were diagnosed with GA1 in all of Scandinavia.2 This indicates that there is a relatively high incidence of GA1 amongst the Palestinian Arab population, reflecting the historical tradition of marriages within extended kindreds, particularly in isolated villages. In most patients in this series, the biochemical diagnosis of GA1 was relatively straightforward after detecting markedly increased excretion of both GA and 3-HGA as well as elevated blood spot glutarylcarnitine. The biochemical diagnosis was more challenging, however, in the two patients (]1 and ]12) with normal GA excretion and normal or only marginally increased blood spot glutarylcarnitine levels. Both had somewhat elevated 3-HGA excretion as a clue to the diagnosis. In the absence of overt glutaric aciduria, however, isolated 3-HGA excretion can easily be overlooked, as the 3-HGA peak in the organic acid chromatogram co-elutes with and may be obscured by the normally more prominent peak of 2-hydroxyglutaric acid. A specific isotope dilution assay for the quantitation of 3-HGA is therefore required.16 Even if recognized, elevated 3-HGA excretion is no longer considered to be pathognomonic of GA1, having also been reported in ketosis,23 SCHAD deficiency24,25 and CPT1 deficiency.26 In this context, our finding of increased urinary glutarylcarnitine excretion, despite normal (patient ]1) or only marginally elevated (patient ]12) blood spot glutarylcarnitine concentration, is of importance. It supports the contention that elevated glutarylcarnitine excretion is a pathognomonic biochemical marker of GA1, with particular application to the subgroup of GA1 patients having normal GA excretion and blood spot glutarylcarnitine levels.6 Mutation analysis of the GCDH gene revealed four previously unreported mutations, including one in-frame trinucleotide insertion and three missense mutations. The pathogenicity of the three novel missense mutations p.Thr341Pro, p.Ans392Ser and p.Met405Val is supported by the observations that they each affect a residue that is conserved in other species and that the p.Asn392Ser mutation involves a residue which is the site of another missense mutation, p.Asn392Asp.27 Furthermore, these mutations were not encountered in 210 control alleles. The other five missense mutations identified in our patients have been previously reported. The p.Ler283Pro, p.Gly390Arg, p.Thr416Ile and p.Ala293Thr mutations were previously identified in Palestinian patients22 and the p.Ala293Thr mutation was also identified in 14 of 43 Spanish GA1 patients.5 The p.Ser119Leu mutation, identified on the paternal allele of the Jewish patient (]12) was previously reported in a Spanish patient.5 GA1 is a genetically heterogeneous disorder with more than 140 mutations having been identified.5,27,28 Many mutations are unique to a single family, but there are some common mutations such as p.Arg402Trp, the most prevalent mutation in Europeans.29 Single founder mutations are responsible for the high incidence of GA1 in specific ethnic groups, for example the p.Ala421Val mutation in the Lancaster Old Order Amish30 or the IVS1+5g4t splicing mutation in the

11 (2007) 81 – 89

Island Lake (Ojibway-Cree) Indians in Central Canada.31,32 Our findings, combined with those of Anikster et al.,22 suggest that a different situation prevails in the Palestinian Arab population. The high incidence of GA1 in this population is a result of multiple mutations, some of which (p.Leu283Pro, p.Ala293Thr, p.Gly390Arg, p.Thr416Ile) have been found in several apparently unrelated families. Similar observations have been made for other recessive disorders in the Palestinian Arab community, such as the neurometabolic disorders metachromatic leukodystrophy33 and non-ketotic hyperglycinemia.34–37 As expected in consanguineous marriages, most mutations were found in homozygosity, but even in this situation compound heterozygosity for two mutations can occur, as in the family of patients ]8 and ]9 in this series. This phenomenon of multiple allelic mutations in the Palestinian community is thought to indicate that recessive mutations are, in fact, not a rare event and can become established when they occur in the context of large consanguineous kindreds, initially being confined to an isolated village but later spreading through the community.38 Determining the molecular basis of GA1 has important practical applications. Mutation analysis is helpful in confirming the diagnosis of GA1 in patients with normal GA excretion, as in patients ]1 and ]12 in this report. Identification of the responsible mutations is also important for enabling carrier detection and prenuptial genetic counseling in communities such as the Palestinian and other Arab populations where there is a high rate of consanguineous marriages.39 Most importantly, prenatal diagnosis of GA1 can be achieved by mutation testing on DNA isolated from CVS tissue, provided that the responsible mutation(s) has been identified in the proband.40 The molecular approach to prenatal diagnosis has several advantages over biochemical approaches. Determination of GA and 3HGA concentrations in amniotic fluid can only be performed in the second trimester, which may be too late to allow termination of pregnancy in certain religious societies. Glutaryl-CoA dehydrogenase activity can be measured in CVS tissue, ideally both directly and in cultured chorionic cells41, but this enzyme assay is not widely available, requires shipment of a labile sample and involves delay for cultivation of chorionic cells. Furthermore, the assay may be subject to error in cases with relatively high residual enzyme activity.42 In contrast, the molecular approach enables rapid, straightforward and reliable prenatal diagnosis of GA1, as illustrated in three families from this series. There are important issues to be considered when counseling families regarding prognosis and prenatal diagnosis. One is the uncertainty regarding the long-term outcome in patients who escape the acute neurological catastrophe of infancy or early childhood, as did patients ]3 and ]9 in this study. They may remain asymptomatic, but there have been reports of a progressive leukoencephalopathy developing in later childhood or even adult life and manifesting with various symptoms including tremor, dyskinesias, ataxia, gait disturbance, headaches and dementia.4,43 Whether treatment of asymptomatic patients detected by family or newborn screening can prevent the late complications of GA1 will only be answered by long-term follow-up studies.

ARTICLE IN PRESS E U R O P E A N J O U R N A L O F PA E D I AT R I C N E U R O L O G Y

Another complicating issue in genetic counseling is the unpredictability of phenotype in individuals with the same GCDH genotype. The clinical heterogeneity in GA1 phenotype observed in this and previous reports is not well understood. Patient ]8 presented in the neonatal period whilst his uncle (patient ]9) with the same genotype was essentially asymptomatic and diagnosed only in adult life. Three of the four patients homozygous for the p.Thr416Ile mutation were severely affected whilst one remained asymptomatic. Asymptomatic and symptomatic siblings homozygous for p.Thr416Ile were also described in the original report of this mutation.22 This phenotypic heterogeneity in patients with the same genotype is consistent with the conclusion that there is no phenotype/genotype correlation in this disorder4,27 and suggests that additional environmental and/or genetic factors determine disease expression.44 An intercurrent illness inducing acute catabolic stress at an age-specific stage of neurosusceptibility is one such factor. It is possible that polymorphisms or mutations at other loci may increase susceptibility, or may confer protection. In this context, the presence of unrelated neurological disorders (mental retardation of unknown etiology (]6), D-2-hydroxyglutaric aciduria (]3) and anencephaly with multiple congenital malformations (]8)) in unaffected siblings in three families may be of significance. Prompt recognition and accurate diagnosis of an encephalopathic episode is essential to minimize the extent of striatal damage and to enable prevention of future recurrences and appropriate genetic counseling to the family. If GA1 can be diagnosed prior to the occurrence of an acute encephalopathic crisis, appropriate measures can be instituted to largely prevent the catastrophic neurological deterioration resulting from acute striatal damage. Early recognition of GA1 is thus the key to minimizing the horrendous morbidity associated with this disorder. Expanded newborn screening using tandem mass spectrometry for detection of glutarylcarnitine can enable presymptomatic detection and preemptive management of affected newborns.45 Nevertheless, glutarylcarnitine levels may be normal in a substantial proportion of patients6 and the diagnosis of GA1 may therefore be missed by newborn screening in some patients.46,47 Furthermore, many countries/states do not have expanded newborn screening programs that include GA1 screening. In these situations, suspicion of and investigation for the possible diagnosis of GA1 remain dependent on the clinical acumen of medical practitioners. In this context, however, our experience indicates that there is a lack of awareness of the typical clinical and neuroimaging features of GA1 amongst general pediatricians, neurosurgeons and radiologists. In only three of the twelve patients in this series was the specific diagnosis of GA1 suspected prior to the referral of samples for general metabolic screening, even though the clinical and neuroimaging features were often typical. This would suggest that GA1 is being under-diagnosed. In these circumstances, there is little likelihood of diagnosing GA1 in the presymptomatic phase and initiating prophylactic therapy. Pediatric neurologists therefore have an important role to play in bringing this disorder to the attention of the broader pediatric community and instructing them as to the key

1 1 (20 07) 81 – 8 9

87

diagnostic clues. Because the majority of newborns affected with GA1 have macrocephaly, general pediatricians and neonatologists should exclude GA1 in newborns and infants with a large head circumference. The finding of bilateral enlarged Sylvian fissures on cranial ultrasound examination or of the typical ‘‘bats wing’’ configuration of the Sylvian fissures on CT or MRI examination should alert neonatologists and radiologists to the possibility of GA1. General and emergency care pediatricians must consider GA1 in parallel with infectious and vascular causes in a previously healthy infant who presents with an acute encephalitis or stroke-like illness. Neurosurgeons and others evaluating patients with head trauma or suspected non-accidental head injury should include GA1 in the differential diagnosis of extracerebral fluid or blood collections.48,49 With such an approach, more patients may benefit from presymptomatic diagnosis and preventive treatment and avoid the devastating consequences of this neurometabolic disorder.

Acknowledgements The authors thank Birthe Roos, Ulbe Holwerda and Erwin E Janssen for excellent metabolite analysis. R E F E R E N C E S

1. Strauss KA, Puffenberger EG, Robinson DL, Morton DH. Type I glutaric aciduria, part 1: natural history of 77 patients. Am J Med Genet C Semin Med Genet 2003;121:38–52. 2. Kyllerman M, Skjeldal O, Christensen E, Hagberg G, Holme E, Lonnquist T, et al. Long-term follow-up, neurological outcome and survival rate in 28 Nordic patients with glutaric aciduria type 1. Eur J Paediatr Neurol 2004;8:121–9. 3. Naughten ER, Mayne PD, Monavari AA, Goodman SI, Sulaiman G, Croke DT. Glutaric aciduria type I: outcome in the Republic of Ireland. J Inherit Metab Dis 2004;27:917–20. 4. Kolker S, Garbade SF, Greenberg CR, Leonard JV, Saudubray JM, Ribes A, et al. Natural history, outcome, and treatment efficacy in children and adults with glutaryl-CoA dehydrogenase deficiency. Pediatr Res 2006;59:840–7. 5. Busquets C, Merinero B, Christensen E, Gelpi JL, Campistol J, Pineda M, et al. Glutaryl-CoA dehydrogenase deficiency in Spain: evidence of two groups of patients, genetically, and biochemically distinct. Pediatr Res 2000;48:315–22. 6. Tortorelli S, Hahn SH, Cowan TM, Brewster TG, Rinaldo P, Matern D. The urinary excretion of glutarylcarnitine is an informative tool in the biochemical diagnosis of glutaric acidemia type I. Mol Genet Metab 2005;84:137–43. 7. Kolker S, Koeller DM, Sauer S, Horster F, Schwab MA, Hoffmann GF, et al. Excitotoxicity and bioenergetics in glutaryl-CoA dehydrogenase deficiency. J Inherit Metab Dis 2004;27:805–12. 8. Varadkar S, Surtees R. Glutaric aciduria type I and kynurenine pathway metabolites: a modified hypothesis. J Inherit Metab Dis 2004;27:835–42. 9. Lehnert W, Sass JO. Glutaconyl-CoA is the main toxic agent in glutaryl-CoA dehydrogenase deficiency (glutaric aciduria type I). Med Hypotheses 2005;65:330–3. 10. Muhlhausen C, Ott N, Chalajour F, Tilki D, Freudenberg F, Shahhossini M, et al. Endothelial effects of 3-hydroxyglutaric acid: implications for glutaric aciduria type I. Pediatr Res 2006;59:196–202.

ARTICLE IN PRESS 88

E U R O P E A N J O U R N A L O F PA E D I AT R I C N E U R O L O G Y

11. Zinnanti WJ, Lazovic J, Wolpert EB, Antonetti DA, Smith MB, Connor JR, et al. A diet-induced mouse model for glutaric aciduria type I. Brain 2006;129:899–910. 12. Funk CB, Prasad AN, Frosk P, Sauer S, Kolker S, Greenberg CR, et al. Neuropathological, biochemical and molecular findings in a glutaric acidemia type 1 cohort. Brain 2005;128:711–22. 13. Monavari AA, Naughten ER. Prevention of cerebral palsy in glutaric aciduria type 1 by dietary management. Arch Dis Child 2000;82:67–70. 14. Korman SH, Salomons GS, Gutman A, Brooks R, Jakobs C. D-2hydroxyglutaric aciduria and glutaric aciduria type 1 in siblings: coincidence, or linked disorders? Neuropediatrics 2004;35:151–6. 15. Struys EA, Korman SH, Salomons GS, Darmin PS, Achouri Y, van Schaftingen E, et al. Mutations in phenotypically mild D-2-hydroxyglutaric aciduria. Ann Neurol 2005;58:626–30. 16. Schor DS, Verhoeven NM, Struys EA, ten Brink HJ, Jakobs C. Quantification of 3-hydroxyglutaric acid in urine, plasma, cerebrospinal fluid and amniotic fluid by stable-isotope dilution negative chemical ionization gas chromatographymass spectrometry. J Chromatogr B Anal Technol Biomed Life Sci 2002;780:199–204. 17. Pitt JJ, Eggington M, Kahler SG. Comprehensive screening of urine samples for inborn errors of metabolism by electrospray tandem mass spectrometry. Clin Chem 2002;48:1970–80. 18. Baric I, Wagner L, Feyh P, Liesert M, Buckel W, Hoffmann GF. Sensitivity and specificity of free and total glutaric acid and 3hydroxyglutaric acid measurements by stable-isotope dilution assays for the diagnosis of glutaric aciduria type I. J Inherit Metab Dis 1999;22:867–81. 19. Twomey EL, Naughten ER, Donoghue VB, Ryan S. Neuroimaging findings in glutaric aciduria type 1. Pediatr Radiol 2003;33:823–30. 20. Neumaier-Probst E, Harting I, Seitz A, Ding C, Kolker S. Neuroradiological findings in glutaric aciduria type I (glutarylCoA dehydrogenase deficiency). J Inherit Metab Dis 2004;27:869–76. 21. Lu¨tcherath V, Waaler PE, Jellum E, Wester K. Children with bilateral temporal arachnoid cysts may have glutaric aciduria type 1 (GAT1); operation without knowing that may be harmful. Acta Neurochir (Wien) 2000;142:1025–30. 22. Anikster Y, Shaag A, Joseph A, Mandel H, Ben Zeev B, Christensen E, et al. Glutaric aciduria type I in the Arab and Jewish communities in Israel. Am J Hum Genet 1996;59:1012–8. 23. Pitt J, Carpenter K, Wilcken B, Boneh A. 3-Hydroxyglutarate excretion is increased in ketotic patients: implications for glutaryl-CoA dehydrogenase deficiency testing. J Inherit Metab Dis 2002;25:83–8. 24. Clayton PT, Eaton S, Aynsley-Green A, Edginton M, Hussain K, Krywawych S, et al. Hyperinsulinism in short-chain L-3hydroxyacyl-CoA dehydrogenase deficiency reveals the importance of beta-oxidation in insulin secretion. J Clin Invest 2001;108:457–65. 25. Molven A, Matre GE, Duran M, Wanders RJ, Rishaug U, Njolstad PR, et al. Familial hyperinsulinemic hypoglycemia caused by a defect in the SCHAD enzyme of mitochondrial fatty acid oxidation. Diabetes 2004;53:221–7. 26. Korman SH, Waterham HR, Gutman A, Jakobs C, Wanders RJ. Novel metabolic and molecular findings in hepatic carnitine palmitoyltransferase I deficiency. Mol Genet Metab 2005;86:337–43. 27. Goodman SI, Stein DE, Schlesinger S, Christensen E, Schwartz M, Greenberg CR, et al. Glutaryl-CoA dehydrogenase mutations in glutaric acidemia (type I): review and report of thirty novel mutations. Hum Mutat 1998;12:141–4. 28. Zschocke J, Quak E, Guldberg P, Hoffmann GF. Mutation analysis in glutaric aciduria type I. J Med Genet 2000;37: 177–81.

11 (2007) 81 – 89

29. Busquets C, Coll MJ, Ribes A. Evidence of a single origin for the most frequent mutation (R402W) causing glutaryl-CoA dehydrogenase deficiency: identification of 3 novel polymorphisms and haplotype definition. Hum Mutat 2000; 15:207. 30. Biery BJ, Stein DE, Morton DH, Goodman SI. Gene structure and mutations of glutaryl-coenzyme A dehydrogenase: impaired association of enzyme subunits that is due to an A421V substitution causes glutaric acidemia type I in the Amish. Am J Hum Genet 1996;59:1006–11. 31. Greenberg CR, Reimer D, Singal R, Triggs-Raine B, Chudley AE, Dilling LA, et al. A G-to-T transversion at the +5 position of intron 1 in the glutaryl CoA dehydrogenase gene is associated with the Island Lake variant of glutaric acidemia type I. Hum Mol Genet 1995;4:493–5. 32. Greenberg CR, Prasad AN, Dilling LA, Thompson JR, Haworth JC, Martin B, et al. Outcome of the first 3-years of a DNA-based neonatal screening program for glutaric acidemia type 1 in Manitoba and northwestern Ontario, Canada. Mol Genet Metab 2002;75:70–8. 33. Heinisch U, Zlotogora J, Kafert S, Gieselmann V. Multiple mutations are responsible for the high frequency of metachromatic leukodystrophy in a small geographic area. Am J Hum Genet 1995;56:51–7. 34. Korman SH, Boneh A, Ichinohe A, Kojima K, Sato K, Ergaz Z, et al. Persistent NKH with transient or absent symptoms and a homozygous GLDC mutation. Ann Neurol 2004;56:139–43. 35. Flusser H, Korman SH, Sato K, Matsubara Y, Galil A, Kure S. Mild glycine encephalopathy (NKH) in a large kindred due to a silent exonic GLDC splice mutation. Neurology 2005;64:1426–30. 36. Boneh A, Korman SH, Sato K, Kanno J, Matsubara Y, Lerer I, et al. A single nucleotide substitution that abolishes the initiator methionine codon of the GLDC gene is prevalent among patients with glycine encephalopathy in Jerusalem. J Hum Genet 2005;50:230–4. 37. Korman SH, Wexler ID, Gutman A, Rolland MO, Kanno J, Kure S. Treatment from birth of nonketotic hyperglycinemia due to a novel GLDC mutation. Ann Neurol 2006;59:411–5. 38. Zlotogora J. Molecular basis of autosomal recessive diseases among the Palestinian Arabs. Am J Med Genet 2002;109:176–82. 39. Zlotogora J, Barges S, Bisharat B, Shalev SA. Genetic disorders among Palestinian Arabs. 4: genetic clinics in the community. Am J Med Genet A 2006;140:1644–6. 40. Busquets C, Coll MJ, Merinero B, Ugarte M, Ruiz MA, Martinez BA, et al. Prenatal molecular diagnosis of glutaric aciduria type I by direct mutation analysis. Prenat Diagn 2000;20:761–4. 41. Christensen E. Prenatal diagnosis of glutaryl-CoA dehydrogenase deficiency: experience using first-trimester chorionic villus sampling. Prenat Diagn 1994;14:333–6. 42. Goodman SI. Prenatal diagnosis of glutaric acidemias. Prenat Diagn 2001;21:1167–8. 43. Bahr O, Mader I, Zschocke J, Dichgans J, Schulz JB. Adult onset glutaric aciduria type I presenting with a leukoencephalopathy. Neurology 2002;59:1802–4. 44. Strauss KA, Morton DH. Type I glutaric aciduria, part 2: a model of acute striatal necrosis. Am J Med Genet C Semin Med Genet 2003;121:53–70. 45. Lindner M, Ho S, Fang-Hoffmann J, Hoffmann GF, Kolker S. Neonatal screening for glutaric aciduria type I: strategies to proceed. J Inherit Metab Dis 2006;29:378–82. 46. Smith WE, Millington DS, Koeberl DD, Lesser PS. Glutaric acidemia, type I, missed by newborn screening in an infant with dystonia following promethazine administration. Pediatrics 2001;107:1184–7. 47. Gallagher RC, Cowan TM, Goodman SI, Enns GM. Glutaryl-CoA dehydrogenase deficiency and newborn screening: retrospective

ARTICLE IN PRESS E U R O P E A N J O U R N A L O F PA E D I AT R I C N E U R O L O G Y

analysis of a low excretor provides further evidence that some cases may be missed. Mol Genet Metab 2005;86: 417–20. 48. Woelfle J, Kreft B, Emons D, Haverkamp F. Subdural hemorrhage as an initial sign of glutaric aciduria type 1: a diagnostic pitfall. Pediatr Radiol 1996;26:779–81.

1 1 (20 07) 81 – 8 9

89

49. Morris AA, Hoffmann GF, Naughten ER, Monavari AA, Collins JE, Leonard JV. Glutaric aciduria and suspected child abuse. Arch Dis Child 1999;80:404–5.