Cerebral Folate Deficiency

YMGME-05016; No. of pages: 4; 4C: Molecular Genetics and Metabolism xxx (2011) xxx–xxx

Contents lists available at ScienceDirect

Molecular Genetics and Metabolism

journal homepage: www.elsevier.com/locate/ymgme

Regular Article Cerebral folate deﬁciency: A neurometabolic syndrome?☆,☆☆

Sarah Mangold a,⁎, Nenad Blau b,c,d, Thomas Opladen e, Robert Steinfeld f, Britta Weßling a, Klaus Zerres g, Martin Häusler a a Department of Pediatrics, University Hospital RWTH Aachen, Germany b Division of Clinical Chemistry and Biochemistry, University Children's Hospital Zürich, Switzerland c Zürich Center for Integrative Human Physiology (ZIHP), Zürich, Switzerland d Research Center for Children (RCC), Zürich, Switzerland e Department of Pediatrics, University Hospital Heidelberg, Germany f Department of Pediatrics, University Hospital Göttingen, Germany g Department of Human Genetics, University Hospital RWTH Aachen, Germany article info abstract

Article history: Background: Cerebral folate deficiency (CFD) is increasingly recognized in various neurological conditions, Received 19 April 2011 raising the question of whether it might represent a clear-cut clinical syndrome. Received in revised form 7 June 2011 Methods: Retrospective analysis of patients with low cerebral spinal fluid (CSF) 5-methyltetrahydrofolate Accepted 7 June 2011 (5MTHF) values was performed. Available online xxxx Results: 58 pediatric patients with low (-2nd to -3rd standard deviation) and 45 patients with very low 5MTHF values (b3rd standard deviation) were identified, including 22 patients with defined underlying neurological Keywords: conditions. The leading symptoms were mental retardation (n=84), motor retardation (n=75), epilepsy Cerebral folate deficiency Antiepileptics (n=53), ataxia (n=44) and pyramidal tract signs (n=37). There was no relationship between 5MTHF levels Mental retardation and the severity of clinical disease, the duration of clinical disease, distinct neurological symptoms and Epilepsy antiepileptic drug treatment, respectively. Genetical analysis for mutations in the folate receptor 1 gene Ataxia proved normal in all 16 children studied. Conclusions: For the majority of patients CFD is not a clear-cut neurometabolic syndrome but the common result of different genetic, metabolic or unknown processes. Nevertheless, CFD may represent a treatable disease-modifying factor which should therefore be addressed in prospective studies. © 2011 Elsevier Inc. All rights reserved.

1. Introduction for the high clinical impact of folate deficiency might be the fact that folate is a cofactor in processes contributing to preserve the genome, During recent years cerebral folate deficiency (CFD), character- to regulate gene expression, to amino acid metabolism, myelin ized by low cerebrospinal fluid (CSF) folates, has emerged to an formation and neurotransmitter synthesis. Therefore, screening for important differential diagnosis in various neurological disorders. low CSF 5-methyltetrahydrofolate (5MTHF) levels has been recom- CFD may be associated with different underlying conditions such as mended for various neurological disorders of unknown origin [8]. 5,10-methylene-tetrahydrofolate reductase, 3-phosphoglycerate de- Based on case series a distinct clinical phenotype of CFD has been hydrogenase, dihydrofolate reductase (DHFR) or dihydropteridine proposed, characterized by normal development until 4–6 months of reductase deficiency, hereditary folate malabsorption, Rett syn- age and evolving agitation, insomnia, psychomotor retardation, drome, Aicardi–Goutières syndrome or mitochondrial disorders deceleration of head growth, ataxia, hypotonia, spasticity, dyskinesia and has also been linked to autistic features [1–7]. One explanation and epilepsy [9]. The underlying etiology causing CFD is not understood. In some cases CFD was shown being related to blocking auto-antibodies to folate receptors [10]. Recently, three patients with ☆ This work was supported in part by the Swiss National Science Foundation grant impaired uptake of folate to the CNS based on a mutation in the folate no. 31003A-119982/2 (to N.B.). ☆☆ All authors disclose any financial relationships or other competing interests that receptor 1 (FOLR1) gene coding for folate receptor alpha, leading to could be perceived as biasing the study. progressive movement disturbance, psychomotor decline, white ⁎ Corresponding author at: Department of Pediatrics, University Hospital RWTH matter disease and epilepsy after an initial period of normal Aachen, Pauwelsstr. 30, 52074 Aachen, Germany. Fax: +49 241 8082484. neuromotor development have been reported [11,12]. This contrasts E-mail addresses: [email protected] (S. Mangold), [email protected] to patients with DHFR deficiency who present with neurologic disease (N. Blau), [email protected] (T. Opladen), fi [email protected] (R. Steinfeld), [email protected] (B. Weßling), combined with systemic MTHF de ciency including megaloblastic [email protected] (K. Zerres), [email protected] (M. Häusler). anemia [13,14] and patients with mutations in the proton-coupled

Please cite this article as: S. Mangold, et al., Cerebral folate deficiency: A neurometabolic syndrome?, Mol. Genet. Metab. (2011), doi:10.1016/ j.ymgme.2011.06.004 2 S. Mangold et al. / Molecular Genetics and Metabolism xxx (2011) xxx–xxx folate transporter (PCFT) in whom systemic folate deficiency is also Table 2 present [15]. Clinical and CSF 5MTHF findings of 22 patients with clear-cut neurological diagnoses. Whereas delineation of a clinical phenotype facilitates diagnosis, Main diagnosis Low CSF Very low CSF Total focusing on few clinical characteristics might exclude patients with 5MTHFa 5MTHFb uncommon symptoms from effective treatment. Rett syndrome 4 2 6 Since 1999 CFD diagnostics has been routinely performed in a large Autism 3 1 4 series of patients treated at RWTH Aachen University Hospital, Mitochondrial disorders 1 1 2 showing otherwise unexplained severe neurological diseases. Some Joubert syndrome 1 0 1 Friedreich ataxia 0 1 1 of these patients have been published previously in order to illustrate Aicardi–Goutières syndrome 0 1 1 specific aspects of CFD [1,2,10]. Data on a larger series of randomly GTP cyclohydrolase I deficiency 1 0 1 selected patients, in contrast, have not yet been reported. Oculomotoric apraxia 1 0 1 3-Phosphoglycerate dehydrogenase 10 1 deficiency 2. Patients and methods Pontocerebellar hypoplasia 0 1 1 Arthrogryposis 0 1 1 Schizophrenia 0 1 1 Clinical charts of patients studied for CFD between 1999 and 2007 Myoadenylate deaminase deficiency 0 1 1 at our hospital by analyzing CSF 5MTHF levels have been analyzed a b b retrospectively. CSF 5MTHF values had been determined by means of -3rd std. dev. 5MTHF -2nd std. dev. b 5MTHFb-3rd std. dev. high-pressure liquid chromatography using electrochemical detection as described previously [16]. The main diagnoses, the biochemical findings and major clinical symptoms were recorded. Among others, this included symptoms considered typical for CFD, such as psycho- symptoms of CFD. Hypothesizing, that patients with severe CFD might motor retardation, microcephaly, ataxia, spasticity, dyskinesia, dys- show more symptoms than patients with moderate CFD we compared tonia and epilepsy. The symptoms were compared with CSF 5MTHF the number of symptoms present in the distinct patients with the concentrations, including only CSF samples obtained prior to any severity of CFD. However, no relationship was found (Fig. 1). folinic acid supplementation. Patients were divided into two groups A long standing CFD might also be related with more severe showing CSF 5MTHF values between age-related -2nd to -3rd clinical disease. Although we cannot rule out that CFD may develop standard deviations (low 5MTHF; moderate CFD) and values age- rapidly, we assumed that CFD should have been present for a longer relatedb-3rd standard deviations (very low 5MTHF; severe CFD) period of time among older children. Therefore, we studied whether (Table 1). Data were compared using the Wilcoxon rank-sum test for children above 5 years of age showing or not showing a distinct independent samples. clinical feature differed with regard to their CSF 5MTHF values. From randomly selected patients genetical analysis of the FOLR1 However, no significant differences were found with regard to any of gene has been performed as reported previously [12]. The analysis the clinical features studied (Table 3). In contrast, older children with was performed in line with the German law on gene diagnostics. white matter changes on MRI even tended to show higher CSF 5MTHF levels than children with normal white matter findings (p=0.0742) 3. Results (Fig. 2). Different drugs, especially antiepileptics, have been discussed to 103 patients with CFD were identified, 64 boys (mean 6±5 years, impair cerebral 5MTHF uptake. 35 of the 103 patients had received range 0–20 years) and 39 girls (mean 5±4 years, range 1–17 years). antiepileptics, including valproic acid in 32 cases. However, patients 58 showed low CSF 5MTHF levels (-3rd std. dev. to -2nd std. dev.) and with or without antiepileptic treatment did not differ with regard to 45 very low levels (below the -3rd std. dev.). 15 of the 45 children their CSF 5MTHF findings. with very low levels showed CSF 5MTHF values lower than 10 nmol/l. A subgroup of 16 children has also been studied for a mutation in In all patients laboratory and clinical signs of systemic folate the FOLR1 gene. Six of them showed low CSF 5MTHF, ten showed very deficiency were absent, ruling out severe enteral folate malabsorp- low CSF 5MTHF levels. No mutations were detected in any of these tion, for example caused by PCFT mutations [15]. theses children. Whereas 22 patients showed distinct underlying clinical diagnoses (Table 2), no clear diagnosis was made in most cases so that the data fi will be further analyzed with regard to the distinct clinical ndings Table 3 (Table 3). Prevalence of distinct clinical findings among patients with low CSF folates. Hypothesizing that symptoms strongly linked to CFD should be “ N Low CSF Very low Ratio very low/low associated with severe CFD, the ratio number of patients with very total 5MTHFa CSF 5MTHFb CSF 5MTHF low/low CSF 5MTHF” was calculated for distinct clinical features. Patient history However, none of the symptoms displayed in Table 3 was associated Preterm infancy 10 5 5 1 with a preponderance of severe CFD. Neonatal cerebral 42 2 1 According to the literature, psychomotor retardation, microceph- hemorrhage aly, ataxia, spasticity, dyskinesia, dystonia and epilepsy may be typical Connatal infection 3 2 1 0.5

Neuroimaging Table 1 CNS malformation 32 19 13 0.7 ﬁ Age-dependent classi cation of CSF 5MTHF levels. White matter changes 16 9 7 0.8 Periventricular leukomalacia 5 4 1 0.25 Age Normal range Low CSF Very low CSF [years] [nmol/l] 5MTHFa [nmol/l] 5MTHFb [nmol/l] Clinical ﬁndings 0–1.99 64–182 34.5–63 b34.5 Mental retardation 84 50 34 0.7 2–4.99 63–111 27–62 b27 Speech disorder 82 48 34 0.7 ≥541–117 22–40 b22 Motor retardation 75 50 25 0.5

a -3rd std. dev.b5MTHFb-2nd std. dev. a -3rd std. dev.b5MTHFb-2nd std. dev. b 5MTHFb-3rd std. dev. b 5MTHFb-3rd std.

Please cite this article as: S. Mangold, et al., Cerebral folate deﬁciency: A neurometabolic syndrome?, Mol. Genet. Metab. (2011), doi:10.1016/ j.ymgme.2011.06.004 S. Mangold et al. / Molecular Genetics and Metabolism xxx (2011) xxx–xxx 3

of CFD, although we cannot rule out that antiepileptic treatment contributed to low CSF 5MTHF concentrations in this distinct subgroup. Considering this clinical heterogeneity it seems difficult to decide whether CFD really contributed to distinct diseases in a substantial fashion and if a search for CFD should generously be recommended for various neurological conditions. The most reliable method to prove the clinical impact of CFD would be clinical amelioration during folinic acid treatment as suggested for some patients with CFD. Folinic acid treatment at dosages of about 1 mg per kg and day led to biochemical improvement with increased CSF 5MTHF concentrations in 68 out of 74 patients (data not shown). Whereas clinical improvement was suggested for distinct patients, this effect was not assessed in a randomized, controlled and prospective fashion. Therefore the clinical impact of folinic acid treatment remains uncertain. Mutations in the FOLR1 gene have recently been shown to cause severe CFD with a severe clinical phenotype and a moderate response to oral folinic acid supplementation [11,12]. The clinical data of the Fig. 1. Comparison of the severity of clinical disease with the severity of CFD (ratio here reported patients differ from the FOLR1-deficient phenotype in number of patients with low/number of patients with very low CSF 5MTHF levels; S=number of symptoms; n=number of patients). A higher number of symptoms was the sense that the later is associated with extremely low or not associated with more severe CFD. undetectable CSF 5MTHF and that increase in CSF 5MTHF requires substantial higher oral folinic acid doses (up to 5 mg/kg per day). In line with these clinical differences we could not detect FOLR1 4. Discussion mutations among 16 patients studied. Although we cannot yet rule out the presence of FOLR1 mutations in other patients studied, further This study reports on a large cohort of patients with CFD, causes other than FOLR1 mutations may explain CFD in the majority of characterized by different, frequently untreatable neurological con- cases. ditions. The clinical heterogeneity is mirrored by the symptoms Most patients with CFD, in turn, do not share a common clinical displayed in Table 3 and by defined underlying diseases known for phenotype so that restriction of CFD diagnostics to few “syndromal” some cases (Table 2). It suggests that CFD frequently represents the symptoms is not advisable. Further studies are required to describe common final pathway of different mechanisms impairing CNS the complete clinical spectrum and to assess the need for therapeutic metabolism rather than a typical syndromal disorder. Our data intervention. Until then, CSF investigation of 5MTHF remains the only support this hypothesis. No patient showed all symptoms considered reliable tool for diagnosing this group of patients. typical for CFD, the number of symptoms present in the distinct patient did not correlate with the severity of CFD and no symptom Acknowledgments was associated with an increased rate of severe CFD when present for a long period of time. Supported in part by the Swiss National Science Foundation grant Uptake of 5MTHF to the CNS has been shown to occur primarily at no. 31003A-119982/2 (to N.B.). the choroid plexus [6]. One assumed secondary cause of CFD may be antiepileptic treatment with valproic acid which might impair uptake References of folate, an hypothesis that is based on the observation that the use of fi antiepileptic drugs (valproic acid, carbamazepine) during pregnancy [1] V.T. Ramaekers, N. Blau, Cerebral folate de ciency, Dev. Med. Child Neurol. 46 (2004) 843–851. increases the risk of neural tube defects and of congenital malforma- [2] V.T. Ramaekers, S.I. Hansen, J. Holm, T. Opladen, J. Senderek, M. Hausler, G. tions in the offspring of women suffering from epilepsy [17–20]. This Heimann, B. Fowler, R. Maiwald, N. Blau, Reduced folate transport to the CNS in – hypothesis was supported by in vitro FOLR1-dependent 5MTHF female Rett patients, Neurology 61 (2003) 506 515. [3] N. Blau, L. Bonafe, I. Krageloh-Mann, B. Thony, L. Kierat, M. Hausler, V. Ramaekers, transport experiments using KB-cell cultures [21]. Among the here Cerebrospinal fluid pterins and folates in Aicardi–Goutieres syndrome: a new reported patients, antiepileptic treatment did not increase the severity phenotype, Neurology 61 (2003) 642–647. [4] A. Garcia-Cazorla, E.V. Quadros, A. Nascimento, M.T. Garcia-Silva, P. Briones, J. Montoya, A. Ormazabal, R. Artuch, J.M. Sequeira, N. Blau, J. Arenas, M. Pineda, V.T. Ramaekers, Mitochondrial diseases associated with cerebral folate deficiency, Neurology 70 (2008) 1360–1362. [5] A. Qiu, M. Jansen, A. Sakaris, S.H. Min, S. Chattopadhyay, E. Tsai, C. Sandoval, R. Zhao, M.H. Akabas, I.D. Goldman, Identification of an intestinal folate transporter and the molecular basis for hereditary folate malabsorption, Cell 127 (2006) 917–928. [6] K. Hyland, J. Shoffner, S.J. Heales, Cerebral folate deficiency, J. Inherit. Metab. Dis. 33 (2010) 563–570. [7] B. Perez-Duenas, A. Ormazabal, C. Toma, B. Torrico, B. Cormand, M. Serrano, C. Sierra, E. De Grandis, M.P. Marfa, A. Garcia-Cazorla, J. Campistol, J.M. Pascual, R. Artuch, Cerebral folate deficiency syndromes in childhood: clinical, analytical, and etiologic aspects, Arch. Neurol. 68 (2011) 615–621. [8] N. Gordon, Cerebral folate deficiency, Dev. Med. Child Neurol. 51 (2009) 180–182. [9] V.T.Ramaekers,M.Hausler,T.Opladen,G.Heimann,N.Blau,Psychomotorretardation, spastic paraplegia, cerebellar ataxia and dyskinesia associated with low 5- methyltetrahydrofolate in cerebrospinal fluid: a novel neurometabolic condition responding to folinic acid substitution, Neuropediatrics 33 (2002) 301–308. [10] V.T. Ramaekers, S.P. Rothenberg, J.M. Sequeira, T. Opladen, N. Blau, E.V. Quadros, J. Selhub, Autoantibodies to folate receptors in the cerebral folate deficiency syndrome, Fig. 2. Comparison of patients with and without white matter changes on magnetic N. Engl. J. Med. 352 (2005) 1985–1991. resonance imaging. The dotted line indicates the lower normal range of CSF 5MTHF [11]H.Cario,H.Bode,K.M.Debatin,T.Opladen,K.Schwarz,Congenitalnullmutationsof values. Older patients (N5 years of age) with white matter changes tend to show higher the FOLR1 gene: a progressive neurologic disease and its treatment, Neurology 73 CSF 5MTHF values than patients without. (2009) 2127–2129.

Please cite this article as: S. Mangold, et al., Cerebral folate deﬁciency: A neurometabolic syndrome?, Mol. Genet. Metab. (2011), doi:10.1016/ j.ymgme.2011.06.004 4 S. Mangold et al. / Molecular Genetics and Metabolism xxx (2011) xxx–xxx

[12] R. Steinfeld, M. Grapp, R. Kraetzner, S. Dreha-Kulaczewski, G. Helms, P. SLC46A1) associated with hereditary folate malabsorption, Mol. Genet. Metab. 103 Dechent, R. Wevers, S. Grosso, J. Gartner, Folate receptor alpha defect causes (2011) 33–37. cerebral folate transport deficiency: a treatable neurodegenerative disorder [16] N.Blau,T.Opladen,Folates,in:N.Blau,M.Duran,K.M.Gibson(Eds.),LaboratoryGuide associated with disturbed myelin metabolism, Am. J. Hum. Genet. 85 (2009) to the Methods in Biochemical Genetics, Springer-Verlag, Berlin Heidelberg, 2008, 354–363. pp. 717–724. [13]S.Banka,H.J.Blom,J.Walter,M.Aziz,J.Urquhart,C.M.Clouthier,G.I.Rice,A.P.de [17] T. Wolff, C.T. Witkop, T. Miller, S.B. Syed, Folic acid supplementation for the prevention Brouwer, E. Hilton, G. Vassallo, A. Will, D.E. Smith, Y.M. Smulders, R.A. Wevers, R. of neural tube defects: an update of the evidence for the U.S. Preventive Services Task Steinfeld, S. Heales, Y.J. Crow, J.N. Pelletier,S.Jones,W.G.Newman,Identification and Force, Ann. Intern. Med. 150 (2009) 632–639. characterization of an inborn error of metabolism caused by dihydrofolate reductase [18] L.V. Dansky, D.S. Rosenblatt, E. Andermann, Mechanisms of teratogenesis: folic acid deficiency, Am. J. Hum. Genet. 88 (2011) 216–225. and antiepileptic therapy, Neurology 42 (1992) 32–42. [14] H. Cario, D.E.C. Smith, H.J. Bloom, N. Blau, H. Bode, K. Holzmann, U. Pannicke, K. [19] B.D. Speidel, S.R. Meadow, Maternal epilepsy and abnormalities of the fetus and Hopfner,E.M.Rump,Z.Ayric,E.Kohne,K.M.Debatin,Y.Smulders,K.Schwarz, newborn, Lancet 2 (1972) 839–843. Dihydrofolate reductase deficiency caused by a homozygous DHFR mutation, leads to [20] M. Mueller-Kueppers, On the problem of fetal damage during pregnancy caused by congenital megalobastic anemia and severe neurologic disease due to cerebral folate intake of anticonvulsants, Acta Paedopsychiatr. 30 (1963) 401–405. deficiency, Am. J. Hum. Genet. 88 (2011) 226–231. [21] T. Opladen, N. Blau, V.T. Ramaekers, Effect of antiepileptic drugs and reactive oxygen [15]D.S.Shin,K.Mahadeo,S.H.Min,N.Diop-Bove,P.Clayton,R.Zhao,I.D.Goldman, species on folate receptor 1 (FOLR1)-dependent 5-methyltetrahydrofolate transport, Identification of novel mutations in the proton-coupled folate transporter (PCFT- Mol. Genet. Metab. 101 (2010) 48–54.

Please cite this article as: S. Mangold, et al., Cerebral folate deﬁciency: A neurometabolic syndrome?, Mol. Genet. Metab. (2011), doi:10.1016/ j.ymgme.2011.06.004