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Treatment of LeschNyhan disease with S-adenosylmethionine: Experience with ve young Malaysians, including a girl
Bee C. Chen a, Shanti Balasubramaniam a,b,, Ivan N. McGown c, J. Patrick ONeill d, Gaik S. Chng a, Wee T. Keng a, Lock H. Ngu a, John A. Duley c,e
b a Department of Genetics, Kuala Lumpur Hospital, Jalan Pahang, 50586 Kuala Lumpur, Malaysia Metabolic Unit, Department of Pediatric and Adolescent Medicine, Princess Margaret Hospital, 6008 Perth, Western Australia c Mater Health Services and Mater Medical Research Institute, Brisbane 4101, Australia d Department of Pediatrics, University of Vermont Genetics Laboratory, Burlington, VT 05405, USA e School of Pharmacy, The University of Queensland, Brisbane 4101, Australia

Received 13 March 2013; received in revised form 24 August 2013; accepted 28 August 2013

Abstract Background: LeschNyhan disease (LND) is a rare X-linked recessive neurogenetic disorder caused by deciency of the purine salvage enzyme hypoxanthine phosphoribosyltransferase (HPRT, EC 2.4.2.8) which is responsible for recycling purine bases into purine nucleotides. Aected individuals have hyperuricemia leading to gout and urolithiasis, accompanied by a characteristic severe neurobehavioural phenotype with compulsive self-mutilation, extrapyramidal motor disturbances and cognitive impairment. Aim: For its theoretical therapeutic potential to replenish the brain purine nucleotide pool, oral supplementation with S-adenosylmethionine (SAMe) was trialed in 5 Malaysian children with LND, comprising 4 related Malay children from 2 families, including an LND girl, and a Chinese Malaysian boy. Results: Dramatic reductions of self-injury and aggressive behaviour, as well as a milder reduction of dystonia, were observed in all 5 patients. Other LND neurological symptoms did not improve during SAMe therapy. Discussion: Molecular mechanisms proposed for LND neuropathology include GTP depletion in the brain leading to impaired dopamine synthesis, dysfunction of G-protein-mediated signal transduction, and defective developmental programming of dopamine neurons. The improvement of our LND patients on SAMe, particularly the hallmark self-injurious behaviour, echoed clinical progress reported with another purine nucleotide depletion disorder, Arts Syndrome, but contrasted lack of benet with the purine disorder adenylosuccinate lyase deciency. This rst report of a trial of SAMe therapy in LND children showed remarkably encouraging results that warrant larger studies. Crown copyright 2013 Published by Elsevier B.V. on behalf of The Japanese Society of Child Neurology. All rights reserved.

Keywords: LeschNyhan disease; HGPRT; HPRT1; Self-injury; Aggression; Dystonia; S-adenosylmethionine

Abbreviations: LND, LeschNyhan disease; HGPRT, hypoxanthine-guanine phosphoribosyltransferase; SAMe, S-adenosyl-methionine; PRPP, 5phosphoribosyl-1-pyrophosphate; HPLC, high performance liquid chromatography; AR, androgen receptor; AICAR, 5-amino-4-imidazole-carboxamide ribotide; IMP, inosine monophosphate; ATIC, AICAR transformylase/IMP cyclohydrolase; GTP, guanosine triphosphate Corresponding author at: Metabolic Unit, Department of Pediatric and Adolescent Medicine, Princess Margaret Hospital, Perth, Australia. Tel.: +61 404302585; fax: +61 893407652. E-mail address: Shanti.Balasubramaniam@health.wa.gov.au (S. Balasubramaniam). 0387-7604/$ - see front matter Crown copyright 2013 Published by Elsevier B.V. on behalf of The Japanese Society of Child Neurology. All rights reserved. http://dx.doi.org/10.1016/j.braindev.2013.08.013

Please cite this article in press as: Chen BC et al. Treatment of LeschNyhan disease with S-adenosylmethionine: Experience with ve young Malaysians, including a girl.. Brain Dev (2013), http://dx.doi.org/10.1016/j.braindev.2013.08.013

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1. Introduction LeschNyhan disease (LND, OMIM 308000) is an inborn error of purine metabolism caused by complete deciency of the enzyme hypoxanthineguanine phosphoribosyltransferase (HGPRT, EC 2.4.2.8) [1]. The worldwide prevalence of HGPRT deciency is estimated at approximately one in 380,000 live births [2]. HGPRT deciency is inherited as a recessive X-linked trait encoded by the HPRT1 gene that is present in all tissues, with the highest levels being found in the basal ganglia [3]. HGPRT deciency can be classied for heuristic reasons based on a continuum spectrum of neurological manifestations and the residual activity of the enzyme into 3 groups: complete HGPRT deciency with classic LND; partial HGPRT deciency with hyperuricemia with neurological disability, and HGPRT-related hyperuricemia with no evident neurological manifestations [4]. The classical LND phenotype is associated with 3 major clinical elements: overproduction of uric acid, neurologic disability and behavioral issues. LND occurs almost exclusively in males, With female carriers usually being asymptomatic: only 7 clinically-aected females are described in the literature, arising from a variety of molecular mechanisms [58]. Uric acid overproduction is present in all HGPRT-decient patients and is associated with urolithiasis, gout and tophi. Megaloblastic

anemia may occur due to ineective erythropoiesis and increased folic acid consumption due to enhanced de novo purine synthesis [9]. HGPRT catalyzes the salvage synthesis of inosine monophosphate (IMP) and guanosine monophosphate (GMP) from the purine bases hypoxanthine and guanine respectively (Fig. 1). HGPRT deciency thus results in decreased reutilization of purine bases that induces a loss of feedback inhibition of de novo purine nucleotide synthesis. Hyperuricemia with hyperuricosuria are the usual biochemical ndings that prompt an enzymatic diagnosis: low or undetectable HGPRT activity in hemolysates, although intact red cell activity is preferable as it facilitates detection of substrate anity defects [10]. Treatment modalities include controlling uric acid overproduction with allopurinol, and management of motor and behavioural manifestations. Allopurinol treatment reduces serum urate and urine uric acid levels; it has not usually been considered to aect behavioral and neurologic symptoms [9]. The lack of precise understanding of the cause of the neurological dysfunction has precluded the development of useful therapies for control the extrapyramidal and behavioural manifestations of the disease. Spasticity and dystonia may be managed with benzodiazepines and GABA inhibitors such as baclofen [12]. Hypotonia is compensated by use of supportive seating and walking

Fig. 1. Purine nucleotide metabolic pathways, including S-adenosylmethionine (SAMe) incorporation. Nucleotides are either synthesised by the de novo pathway (which is low or absent in brain), or are salvaged via HGPRT (6) or adenine phosphoribosyl-transferase (7). Mononucleotides and nucleosides derived from DNA occur in the deoxy- form until catabolised to their bases (hypoxanthine and guanine) by nucleotidase and phosphorylase. The brain is dependent upon HGPRT, particularly for the salvage of GTP which is used to regulate G-proteins and synthesise dopamine. SAMe can cross the bloodbrain barrier and is converted to adenosine following utilisation by methyltransferases and Sadenosylhomocysteine hydrolase (1); this releases adenosine, which is salvaged by adenosine kinase (2), and adenine, which is salvaged by adenine phosphoribosyltransferase (7), to form AMP, which can then be converted to either ATP via nucleotide kinases (3) or to GTP via AMP deaminase (4), IMP dehydrogenase/GMP synthase (5) and nucleotide kinases (3). Figure reproduced with permission of author JAD [11].

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frames. Self-injurious behaviour is managed with a combination of physical restraints, behavioral and pharmaceutical treatments. However, few treatments have proven consistently helpful in ameliorating this devastating feature. A single report describing the dramatic reduction of self-harming in a 43 year old man with LND following oral administration of S-adenosylmethionine (SAMe) [13] and the subsequent anecdotal description of its use in a LND child has showed promising theoretical therapeutic potential of this metabolic supplement [14]. We describe here 5 Malaysian children with LND in whom we had initial diculties in mitigating the severity of their neurological features and their subsequent response to treatment with SAMe. 2. Patients and methods 2.1. Patients A total of 5 Malaysian children with LND, four maternally related Malay cousins including a girl (Fig. 2), were recruited from the outpatient clinic at the Department of Genetics, Kuala Lumpur Hospital, Malaysia. This study to administer SAMe on a trial basis was approved by the Malaysian Human Research Ethics Committee. Informed consent for performing the mutational analysis and studying the therapeutic potential of SAMe were obtained from the parents all subjects following the approved procedures. LND was diagnosed in all 5 patients based on the clinical features of developmental delay, extrapyramidal symptoms, in particular dystonia and the characteristic aggressive, self-injurious behavior; biochemical parameters including hyperuricemia, hyperuricosuria associated with marked elevation of urinary hypoxanthine and xanthine (Table 1); enzymatic assays of HGPRT activity

which was undetectable; and molecular analysis of the HPRT1 gene. The oldest patient in this cohort, patient 1, was a 15 year old Chinese boy rst brought to medical attention at 7 months of age when he presented with hypotonia and global developmental delay with extreme head lag and inability to roll over. At 1 year 4 months of age he was noted to have spasticity and dystonia for which Sinemete (L-dopa) and later baclofen were used without signicant benets. He was referred to the genetics clinic at 3.6 years by which time he had displayed signs of self-mutilation including biting of his lower lip and ngers. Initial biochemical investigations had revealed severe hyperuricemia and hyperuricosuria suggestive of LND and the diagnosis was subsequently conrmed by erythrocyte HGPRT assay which were undetectable. Genetic testing has revealed a novel genomic deletion including exons 1, 2 and 3 and intervening sequences, which resulted in no HPRT1 mRNA. The carrier status of the mother at the time of submission has not been determined. Allopurinol was commenced at 20 mg/kg/day (Table 2) and he was placed on a purine restricted diet: this proved helpful in reducing the uric acid levels however we faced continuous problems managing his selfharming, aggressive behaviour despite having attempted several drugs including diazepam and carbamazepine. When reviewed at age 12 years had a large number of self-inicted wounds: his lower lip had been partially chewed away despite having dental plates and other physical restraints and he had bitten through most of his ngers. He had numerous other partially healing or healed wounds on his forehead sustained from repeated head banging. He was highly agitated and displayed abusive behaviour towards the medical sta, spitting, swearing and kicking despite being restrained in a wheel chair. Patient 1 was the rst of the cohort to be treated

Fig. 2. Malay family pedigree showing Patients 25: patient 2 (II-1), patient 3-(II-4), patient 4 (II-9) and patient 5 (II-10). The asymptomatic mothers (I-5 and I-8) of these patients are sisters. A maternal uncle, who was globally delayed, had passed away at ve years of age without being investigated for his underlying condition. Both sets of parents are well and the maternal grandmother was asymptomatic. There is no history of renal disease, gout or associated neurological features among the remaining seven maternal siblings.

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Table 1 Patient biochemical ndings. Values are prior to allopurinol (Allo) unless otherwise noted. Age at assessment: Patient 1: 3 y 6 m; Patient 2: 5 y 6 m; Patient 3: 6 m; Patient 4: 1 y 10 m Patient 5: 2 d. Patient Serum urate (umol/L) 529 680 628 601 123 755 160 ranges 120320 220320 250350 Urine urate/ Creat (mol/mol) 1.7 2.3 2.0 4.6 0.4 5.0 3.6 0.361.75 0.240.93 0.170.64 Hyp/Creat (mmol/mol) 559 642 951 106 2100 67 nd 048 249 040 Xan/Creat (mmol/mol) 575 708 752 38 1690 49 nd 045 238 239 Oxypur/Creat (mol/mol) 2.81 3.65 3.70 4.74 4.20 5.15 Erythrocyte HGPRT Assay 80130 nmol/min/mm Hb Undetectable Undetectable Undetectable Undetectable Undetectable Undetectable 0.361.85 0.241.02 0.170.72 HPRT1 mutational analysis and genotyping c.1-?_318+?del (IVS8-1G>A) Hemizygote (IVS8-1G>A) Heterozygote (IVS8-1G>A) Hemizygote (IVS8-1G>A) Hemizygote (IVS8-1G>A) Hemizygote

1 2 3 4 (+Allo) 5 (+Allo) Reference <2 y 210 y >10 y

Abbreviations: nd not determined; Creat creatinine; Hyp hypoxanthine; Xan xanthine; Oxypur Oxypurines (urate + hypoxanthine + xanthine); y years; m months; d days.

Table 2 Patient details including allopurinol and SAMe therapies. Clinical improvement was estimated by parents. Patient 1 2 3 4 5 Age at diagnosis 3 y6 mo 5 y6 mo 10 mo 1 y10 mo 2d Onset of self-injury 3+ y 6+ y 16 mo 2y Nil Age allopurinol started 3 y6 mo 5 y8 mo 10 mo 1 y10 mo 2d Age SAMe started 12 y 10 y 4 y1 mo 2 y1 mo 1 mo Allopurinol dose mg/kg/day 20 13 18 22 11 SAMe dose mg/kg/day 22* 21* 33 38** 26 Self-injury improvement 6070% Self-injury ceased 5060% 7080% Absent at 1 y Dystonia improve-ment 2030% 3040% 2030% 30% Mild dystonia

Abbreviations: y years; mo months; d days. * SAMe 200 mg BD, later increased. ** SAMe 150 mg BD.

with SAMe, which was commenced at a dose of 22 mg/ kg/day. SAMe was sourced from Quality Supplements & Vitamins Inc (Fort Lauderdale, USA). The other 4 patients were from 2 maternally related kindreds (Fig. 2). The proband (patient 2-II 1) aged 13 years of age was born after an uncomplicated pregnancy and delivery. Initial concerns arose at 67 months of age because of delayed milestones. Upon presentation he displayed signicant action dystonia superimposed on a baseline hypotonia. Oral clonazepam and later L-dopa were prescribed for his dystonia however these failed to provide much relieve. He was rst referred to the Genetics Department, Kuala Lumpur Hospital at 5 years 6 months of age with progressive psychomotor retardation and severe limb spasticity, dystonia and failure to thrive (weight <3rd percentile). A brain MRI had showed bilateral fronto-temporal atrophy. Biochemical investigations showed gross hyperuricosuria and hyperuricemia (Table 2), and a diagnosis of LND was conrmed by undetectable HGPRT haemolysate activity. Genotyping showed him to be hemizygous for a previously described HPRT1 intronic splice site mutation c.610-1G>A (IVS8-1G>A) [20].

Oral allopurinol was commenced 2 months later, with a purine-restricted diet. Oral baclofen (2 mg tds) produced little improvement of his dystonia. At 6 years and 8 months of age he developed self-injurious behavior manifested by biting his lower lip and ngers. A renal ultrasound performed then showed bilateral medullary nephrocalcinosis. SAMe was commenced 21 mg/ kg/day at the age of 10 years (Table 2). Patient 3 (II-2), is the 7 year old younger sister of Patient 1. She was investigated at 6 months of age after the conrmation of the diagnosis of LND in her brother. Her parents retrospectively reported noting orange crystals in her diapers. Erythrocyte HGPRT assay was found to have zero activity, conrming the diagnosis. The HUMARA gene showed skewed x-inactivation of >95% of wildtype HPRT1 alleles in peripheral blood cells. She was placed on a purine-restricted diet and had allopurinol (18 mg/kg/day) and oral baclofen (2.5 mg bd) commenced at age 10 months and 15 months, respectively. By 16 months of age however she had developed compulsive self- harm (biting her ngers, lip and tongue). Medullary nephrocalcinosis were noted at 2 years of age. She failed to thrive, was globally

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delayed and had signicant dystonia by 3 years 6 months of age. SAMe (33 mg/kg/day) was prescribed at 4 years 1 month of age. Patient 4 (II-9) is the 3.5 year old rst-cousin on the maternal side of the above 2 patients. At presentation, his mother was 36+ weeks pregnant with patient 5 (II10), his younger aected brother. Patient 4 was noted to be globally delayed since early infancy with persistent head lag initially observed at 3 months old. When he rst presented to us at 1 year 10 months of age he clearly displayed signicant dystonia and limb hypertonicity. Diagnosis of LND was conrmed by undetectable erythrocyte HGPRT activity and allopurinol started at 22 mg/kg.day. Oral baclofen (1 mg tds) was added and he was placed on a purine restricted diet. Genetic testing has showed that he was hemizygous for the same HPRT1 mutation as his cousins. When reviewed at 24 months of age he had begun showing self-mutilation by frequently biting his thumbs and ngers, and he was prescribed SAMe. Patient 5 (II-10) was pre-emptively admitted to our special care nursery soon after delivery. He was born well and healthy at full term however at day 2 of life his diapers were noted to show orange sandy crystals. Laboratory investigations revealed severe hyperuricemia and hyperuricosuria (Table 1), but the urinary oxypurine bases hypoxanthine and xanthine were only mildly raised. He was commenced on allopurinol (11.5 mg/ kg/day) on day 3 of life Oral SAMe (26 mg/kg/day) was commenced at 4 weeks of age once the erythrocyte HGPRT assay results were available conrming undetectable activity. Genotyping revealed he was hemizygous for the family mutation in HPRT1. 2.2. Biochemical and genetic analyses 2.2.1. Plasma and urinary uric acid and purine metabolites Plasma and urinary uric acid were determined enzymatically by uricase and urine creatinine by the modied Jae (alkaline-picrate) method, using an auto-analyser. Purine metabolites (hypoxanthine and xanthine) were analyzed by reversed phase HPLC, modied from the method of Simmonds and coworkers [10], using an Agilent 1200 HPLC system including photodiode array detection (Agilent Technologies, Selangor, Malaysia). 2.2.2. Erythrocyte HGPRT activity HGPRT activity in erythrocyte lysates was assayed by the method of Simmonds and coworkers [10] with a reference range of 80130 nmol/min/mg Hb. 2.2.3. Molecular analyses of the HPRT1 gene Molecular analyses of the HPRT1 gene were performed on genomic DNA samples isolated from peripheral blood leukocytes. The 50 UTR, coding regions, and

intron/exon boundaries of the gene were amplied using previously described methods [15]. The PCR products were puried using a Roche HighPure purication kit followed by bi-directional sequencing using an Applied Biosystems Big Dye Terminator v3.1 cycle sequencing kit (Life Technologies Australia P/L, Mulgrave, Australia), according to the manufacturers instructions. Sequences were read on an Applied Biosystems 3130.x1 Genetic Analyzer. 2.2.4. X-inactivation studies For X inactivation studies the human androgenreceptor (HUMARA) gene was analysed as previously described [16], using DNA from peripheral blood. 3. Results Oral supplementation with SAMe surprisingly resulted in dramatic reductions of self-injury and aggressive behaviour, as well as a milder reduction of dystonia, in all 5 patients. There appeared to be a prominent temporal association between the introduction of SAMe and the improvement of symptoms. A similar response had not been observed with the numerous other medications these patients had been on. When patient 1 was reviewed a year after the introduction of SAMe, he had interestingly displayed a much calmer behavior with a sustained reduction of aggression and self-harming of about 6070%. His mother also noted an improvement in his dystonic posturing, which had decreased by 20%. Particularly notable was his mothers expressions of gratitude for the remarkable improvement of his temperament and his reduction of self-injury, which were only observed after his beginning SAMe supplementation. Similarly, remarkable reductions of self-mutilation and to a lesser degree of dystonia were recorded by parents of the other 4 patients (Table 2). Patient 5, the youngest of this group, was commenced on both allopurinol and SAMe soon after birth. When reviewed at 12 weeks, he had good head control, normal tone and reexes, with no obvious dystonia noted. At 9 months of age his mother observed some dystonia, but this was signicantly milder in comparison to his elder brother in whom the onset of dystonia was earlier and more severe. He was mildly hypotonic and delayed in his gross motor milestones, being unable to sit unsupported at that stage, however had appeared to be more advanced at age related developmental assessments compared to his elder brother. Interestingly, the mother of patients 4 and 5 had specically requested for treatment with SAMe in both her aected children as she had received encouraging feedback from her sister (mother of patients 2 and 3) who had described the signicant alleviation of aggression and self-harm in her two aected children.

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4. Discussion More than ve decades have passed since the original description by Lesch and Nyhan (1964) of this devastating disease [1], however much remains to be learned regarding the pathophysiology of its neurobehavioural dysfunction. Despite ubiquitous expression of HPRT, the consequences of HPRT deciency in humans predominate in the nervous system. Adjunctive medications that are useful for attenuating anxiety and stress which are known to exacerbate the self-injurious behaviour in LND include benzodiazepines and carbamazapine. The antiepileptic drug gabapentin has been suggested in one report to have improved the self-injurious behavior without any associated side eects [17], but controlled trials have not been conducted. Risperidone, a nonselective antagonist of both 5-HT2A and dopamine D2 receptors, has been found to reduce self-injurious behavior in another report [18]. Other treatments under investigation include local injections of botulinum toxin into the bilateral masseters or into the facial muscles to prevent tongue and lip biting [19] and deep brain stimulation in globus pallidus [20] but the ecacy and safety of these therapies need to be demonstrated in the longterm management of these patients. The extrapyramidal motor syndrome with involuntary movements and dystonia, and behavioral disturbance has been linked with dysfunction of the basal ganglia and particularly its dopaminergic pathways. Neurochemical analyses of post-mortem brain tissues [21] as well as neuroimaging studies [22] have shown that HGPRT deciency is associated with a marked loss of basal ganglia dopamine and dopamine-related functions. LND is associated with low purine nucleotide levels, particularly guanosine triphosphate (GTP), which is essential for the rst step of pterin and dopamine synthesis by GTP cyclohydrolase. However, pharmacologic treatments have not taken into account the associated nucleotide depletion and dopamine hypersensitivity resulting from HGPRT deciency [9]. More recently, a defect in developmental programming of dopaminergic neurons, mediated through engrailed-1 and -2 (transcription factors known to play a key role in the specication and survival of dopamine neurons), has been proposed to be implicated in the neurological manifestations of LNS [23]. Finally, the alteration of nucleotide concentrations has been postulated to cause changes in G-protein-mediated signal transduction [24]. Self-mutilation and aggression are the cardinal, devastating features of LND that have remained particularly dicult to manage despite various approaches to limit the destructive pattern of behavior, including pharmacological and behavioral modication techniques. No medication has been found to eectively control the extrapyramidal manifestations of the disease [9]. Dopamine replacement therapy has been reported in a

few uncontrolled trials with variable results, with most treated patients displaying intolerable side-eects [25]. This could be explained by the dopamine decit in LND, causing up regulation of dopamine receptors (demonstrated by immunohistochemical methods in the post-mortem analysis of the putamen and the caudate nucleus of two LND patients), resulting in subsequent dopaminergic hypersensitivity [26]. Our interest in the potential therapeutic benet of SAMe, a methyl-nucleotide dietary supplement, for LND arose from a report of SAMe administration to a 43 year old LND patient to treat liver dysfunction which arose from one of the medications he was receiving. Surprisingly, in addition to the prompt improvement of his liver enzymes, an amelioration of selfinjury occurred [13]. A brief mention of the benet of SAMe for LND has also appeared in one other report [14]. The most conspicuous eect in our patients described here was the temporal association of the dramatic reduction of aggression and self-harm with the introduction of SAMe. None of the other medications used in these patients had produced similar responses. SAMe is a precursor for 3 important pathways, transmethylation, transsulfuration and aminopropylation [27]. But metabolism of SAMe also provides an adenosyl moiety, which can be salvaged to form adenosine mononucleotide, AMP, by both adenine phosphoribosyltransferase and adenosine kinase [28]. This AMP is a substrate for the nucleotide interconversion pathways to form other ATP as well as guanosine nucleotides such as GTP (Fig. 1). This potential of SAMe to supplement endogenous nucleotides may thus account for the improvement in the neuropathology of our LND patients. This is echoed by the benecial eects reported for another purine nucleotide depletion disorder, Arts Syndrome [29]. It is known that, unlike other nucleotides administered orally, SAMe is eciently taken up into the bloodstream and crosses the bloodbrain barrier, as evidenced by the ecacy of SAMe for treatment of major depressive disorders that clinical studies have described [30]. In LND, we postulate that oral SAMe supplementation could perhaps alleviate the extrapyramidal symptoms of dopamine hypersensitivity by two mechanisms: (a) increasing GTP synthesis that is essential for pterin and dopamine synthesis, and (b) enhancing the synthesis of catechol-O-methyltransferase (COMT) which is involved in the inactivation of dopamine post-synaptically. Hence the exacerbation of symptoms previously reported with dopamine replacement therapy was not observed with SAMe. This open-label clinical trial utilizing SAMe in the 5 LND patients and was largely based on a previous report on its use with a single LND patient [13] and its ecacy for treating 2 patients with Arts syndrome [29]: based on this oral SAMe supplementation was targeted at a dose of 2030 mg/kg/day, although one

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patients dose exceeded this range. Further dose escalation has not been attempted. SAMe should only be commenced in patients with diagnostic conrmation of LND or in high risk siblings of conrmed LND patients who have biochemical evidence of purine overproduction. Biochemical monitoring was not addressed in our study, due to the limited resources of the laboratory service, however we hope our experience will encourage further studies of SAMe therapy for LND, where monitoring of plasma SAMe levels may be included. Treatment of a single patient with adenylosuccinate lyase deciency with 35 mg/kg/day SAMe was accompanied by plasma assays of amino acids, folate levels, total homocysteine and vitamin B12 levels, which showed no detrimental increases in plasma urate or homocysteine concentrations [31]. Treatment endpoints aimed for in our study included behavioural outcome assessments; self-mutilation, aggression, irritability as measured by percent positive mood rating which describes the percentage of waking hours during which positive mood is experienced [13]; and neurological manifestations of dystonia, chore-athetoid movements. We would suggest that 6 months may be a reasonable trial period for SAMe, unless there are signs of intolerance. Treatment of our patients also drew our attention to an interesting but concerning report relevant to LND therapy describing an association between the onset of self-injury and allopurinol therapy. LND patients excrete high levels of the unusual allopurinol metabolite oxypurinol-riboside, which coincides with abnormal accumulation of the pyrimidine metabolite orotidine [32]. This derangement of pyrimidine nucleotide metabolism in LND patients during allopurinol therapy occurs because oxypurinol-ribotide inhibits orotidine50 -monophosphate decarboxylase, the second reaction of UMP synthase, and normally causes an increased excretion of orotic acid this is the basis of the allopurinol load test for some urea cycle defects. The absence of HGPRT results in high cellular levels of phosphoribosyl-pyrophosphate, which increases conversion of orotic acid to its nucleotide, orotidine monophosphate [32]. Intriguingly, we observed in 3 out of our 5 patients the initiation of allopurinol therapy was followed by the onset of self-injury. This was particularly obvious in Patient 2, who had not begun allopurinol therapy until 6 years of age, but when reviewed 12 months later he had begun exhibiting self-harming behaviour. Allopurinol was given to Patient 3 from 10 months age, and within 8 months had developed self-injury. Two months after Patient 4 was commenced on allopurinol he too demonstrated self-injurious behavior. The association of onset of self-injury and allopurinol therapy could be purely coincidental or alternatively a mere manifestation of the natural history of the disease with the initial appearance of motor dysfunction, followed

by behavioural abnormalities. he potential for neurotoxicity by allopurinol in LND should however be borne in mind. We hypothesize that the derangement of nucleotides produced by allopurinol in LND patients may exacerbate the nucleotide depletion syndrome produced by HGPRT deciency, combined with the presence of the neurotoxic nucleotide AICAR-ribotide which accumulates in LND patients [11]. An alternative option should be adopted for lowering urate production, and one such candidate is the xanthine oxidase inhibitor febuxostat. In summary, these ve LND patients are the rst reported in Malaysia and include a rare case of a severely aected female. We found that SAMe therapy produced a consistent and dramatic improvement in self-harming behavior and to a lesser extent in the control of dystonia in our patients. We propose that SAMe could theoretically replenish brain guanosine nucleotides thus increasing dopamine synthesis, while simultaneously activating COMT turnover of postsynaptic dopamine thereby decreasing dopamine hypersensitivity. We suggest that our encouraging ndings with SAMe therapy be explored further to provide insights into the neuropathology of the disorder and the underlying mechanism mediating its eects.

Acknowledgments We would like to express our gratitude to the Director General of Health, Malaysia for approving the use of SAME and for allowing the publication of this paper. This work was supported by a grant from the Ministry of Health, Malaysia (MRG-2006-35). Dr. Winnie Ong and Dr. Muzhirah Aisha are thanked for assisting with patient treatment and management, and Norsiah Bt Desa and Nor Azimah Abd Azize for mutation analysis of Patient 5. JON thanks the LeschNyhan Syndrome Childrens Research Foundation for generous support of HPRT1 mutation analyses. We also thank the parents of the patients who had entrusted our clinical team with the management of their children with the o-license use of SAMe. LHN is acknowledged as co-senior author. References
[1] Lesch M, Nyhan WL. A familial disorder of uric acid metabolism and central nervous system function. Am J Med 1964;36:56170. [2] Bertelli M, Randi D, Micheli V, Gallo S, Andrighetto G, Parmigiani P, et al. Molecular basis of hypoxanthine-guanine phosphoribosyltransferase deciency in Italian LeschNyhan patients: identication of nine novel mutations. J Inherit Metab Dis 2004;27:76773. [3] Patel PI, Framson PE, Caskey CT, Chinault AC. Fine structure of the human hypoxanthine phosphoribosyltransferase gene. Mol Cell Biol 1986;6:393403. [4] Jinnah HA, Freedmann T. LeschNyhan disease and its variants. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The

Please cite this article in press as: Chen BC et al. Treatment of LeschNyhan disease with S-adenosylmethionine: Experience with ve young Malaysians, including a girl.. Brain Dev (2013), http://dx.doi.org/10.1016/j.braindev.2013.08.013

B.C. Chen et al. / Brain & Development xxx (2013) xxxxxx metabolic and molecular bases of inherited disease. New York: McGraw-Hill. p. 253770. De Gregorio L, Nyhan WL, Seran E, Chamoles NA. An unexpected aected female patient in a classical LeschNyhan family. Mol Genet Metab 2000;69:2638. Jinnah HA, De Gregorio L, Harris JC, Nyhan WL, ONeill JP. The spectrum of inherited mutations causing HPRT deciency: 75 new cases and a review of 196 previously reported cases. Mutat Res 2000;463:30926. De Gregorio L, Jinnah HA, Harris JC, Nyhan WL, Schretlen DJ, Trombley LM, et al. LeschNyhan disease in a female with a clinically normal monozygotic twin. Mol Genet Metab 2005;85:707. Rinat C, Zoref-Shani E, Ben-Neriah Z, Bromberg Y, BeckerCohen R, Feinstein S, et al. Molecular, biochemical, and genetic characterization of a female patient with LeschNyhan disease. Mol Genet Metab 2006;87:24952. Torres RJ, Puig JG. LeschNyhan syndrome. Drugs Fut 2010;35:4217. Simmonds HA, Duley JA, Davies PM. Analysis of purines and pyrimidines in blood, urine and other physiological uids. In: Hommes FA, editor. Techniques in diagnostic human biochemical genetics. Wiley-Liss: New York; 1990. p. 397424. Duley JA, Christodoulou J, de Brouwer AP. The PRPP synthetase spectrum: what does it demonstrate about nucleotide syndromes? Nucleosides Nucleotides Nucleic Acids 2011;30:112939. Jinnah HA, Visser JE, Harris JC, Verdu A, Larovere L, CeballosPicot I, et al. Delineation of the motor disorder of LeschNyhan disease. Brain 2006;129:120117. Glick N. Dramatic reduction in self-injury in LeschNyhan disease following S-adenosylmethionine administration. J Inherit Metab Dis 2006;29:687. McCarthy GT, Green EM, Ogunbona O, Simmonds HA, Fairbanks L, Pountney T, et al. A population study of Lesch Nyhan disease in the UK. Dev Med Child Neurol 2011;53:349. Gibbs R, Nguyen PN, Edwards A, Civitello AB, Caskey CT. Multiplex DNA deletion detection and exon sequencing of the hypoxanthine phosphoribosyltransferase gene in LeschNyhan families. Genomics 1990;7:23544. Allen CR, Zoghbi HY, Moseley AB, Rosenblatt JW, Belmont JW. Methylation of HpaII and HhaI sites near the polymorphic CAG repeat in the human androgen-receptor gene correlates with X chromosome inactivation. Am J Hum Genet 1992;51:122939. McManaman J, Tam DA. Gabapentin for self-injurious behavior in LeschNyhan syndrome. Pediatr Neurol 1999;20:3812. Allen SM, Rice SN. Risperidone antagonism of self-mutilation in a LeschNyhan patient. Prog Neuropsychopharmacol Biol Psychiatry 1996;20:793800. Dabrowski E, Smathers SA, Ralstrom CS, Nigro MA, Leleszi JP. Botulinum toxin as a novel treatment for self-mutilation in Lesch Nyhan syndrome. Dev Med Child Neurol 2005;47:6369. [20] Pralong E, Pollo C, Coubes P, Bloch J, Roulet E, Tetreault MH, et al. Electrophysiological characteristics of limbic and motor globus pallidus internus (GPI) neurons in two cases of Lesch Nyhan syndrome. Neurophysiol Clin 2005;35:16873. [21] Lloyd KG, Hornykiewicz O, Davidson L, Shannak K, Farley I, Goldstein M, et al. Biochemical evidence of dysfunction of brain neurotransmitters in the LeschNyhan syndrome. N Engl J Med 1981;305:110611. [22] Ernst M, Zametkin AJ, Matochik JA, Pascualvaca D, Jons PH, Hardy K, et al. Presynaptic dopaminergic decits in LeschNyhan disease. N Engl J Med 1996;334:156872. [23] Ceballos-Picot I, Mockel L, Potier MC, Dauphinot L, Shirley TL, Torero-Ibad R, et al. Hypoxanthine-guanine phosphoribosyltransferase regulates early developmental programming of dopamine neurons: implications for LeschNyhan disease pathogenesis. Hum Mol Genet 2009;18:231727. [24] Pinto CS, Seifert R. Decreased GTP-stimulated adenylylcyclase activity in HPRT-decient human and mouse broblast and rat B103 neuroblastoma cell membranes. J Neurochem 2006;96:4549. rez-Duen bal A, Artuch R, Campistol [25] Serrano M, Pe as B, Ormaza J, Torres RJ, et al. Levodopa therapy in a LeschNyhan disease patient: pathological, biochemical, neuroimaging, and therapeutic remarks. Mov Disord 2008;23:1297300. a MG, Puig JG, Torres RJ. Abnormal adenosine and [26] Garc dopamine receptor expression in lymphocytes of LeschNyhan patients. Brain Behav Immun 2009;23:112531. [27] Baric I. Inherited disorders in the conversion of methionine to homocysteine. J Inherit Metab Dis 2009;32:45971. [28] Montero C, Smolenski RT, Duley JA, Simmonds HA. Sadenosylmethionine increases erythrocyte ATP in vitro by a route independent of adenosine kinase. Biochem Pharmacol 1990;40:261723. [29] de Brouwer APM, Williams KL, Duley JA, van Kuilenburg ABP, Nabuurs SB, Egmont-Petersen M, et al. Arts syndrome is caused by loss-of-function mutations in PRPS1. Am J Hum Genet 2007;81:50718. [30] Bottiglieri T. S-Adenosyl-L-methionine (SAMe): from the bench to the bedsidemolecular basis of a pleiotrophic molecule. Am J Clin Nutr 2002;76:1151S7S. [31] van Werkhoven M, Duley J, McGown I, Munce T, Freeman J, Pitt J. Early diagnosis of adenylosuccinate lyase deciency with a high throughput screening method and a trial of oral Sadenosylmethionine. Dev Med Child Neurol 2013. http://dx.doi.org/ 10.1111/dmcn.12244. [Epub ahead of print]. [32] Simmonds HA, Reiter S, Davies PM, Cameron JS. Orotidine accumulation in human erythrocytes during allopurinol therapy: association with high urinary oxypurinol-7-riboside concentrations in renal failure and in the LeschNyhan syndrome. Clin Sci Lond 1991;80:1917.

[5]

[6]

[7]

[8]

[9] [10]

[11]

[12]

[13]

[14]

[15]

[16]

[17] [18]

[19]

Please cite this article in press as: Chen BC et al. Treatment of LeschNyhan disease with S-adenosylmethionine: Experience with ve young Malaysians, including a girl.. Brain Dev (2013), http://dx.doi.org/10.1016/j.braindev.2013.08.013