Clinica Chimica Acta 440 (2015) 201–204
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Case report
NMR-based urinalysis for rapid diagnosis of β-ureidopropionase deficiency in a patient with Dravet syndrome Ching-Wan Lam a,⁎, Chun-Yiu Law a, Ka-Fei Leung a, Chi-Kong Lai b, Sammy Pak-lam Chen b, Bosco Chan c, Kwok-yin Chan c, Yuet-ping Yuen b, Chloe Miu Mak b, Albert Yan-wo Chan b a b c
Department of Pathology, The University of Hong Kong, Hong Kong, China Department of Pathology, Princess Margaret Hospital, Hong Kong, China Department of Paediatrics and Adolescent Medicine, Princess Margaret Hospital, Hong Kong, China
a r t i c l e
i n f o
Article history: Received 13 October 2014 Received in revised form 17 October 2014 Accepted 21 October 2014 Available online 25 October 2014 Keywords: NMR spectroscopy β-Ureidopropionase deficiency Dravet syndrome Dual molecular diagnoses
a b s t r a c t Background: Beta-ureidopropionase deficiency is a rare inborn error of metabolism (IEM) affecting pyrimidine metabolism. To-date, about 30 genetically confirmed cases had been reported. The clinical phenotypes of this condition are variable; some patients were asymptomatic while some may present with developmental delay or autistic features. In severe cases, patients may present with profound neurological deficit including hypotonia, seizures and mental retardation. Using NMR-based urinalysis, this condition can be rapidly diagnosed within 15 min. Case: An 11-month-old Chinese boy had dual molecular diagnoses, β-ureidopropionase deficiency and Dravet syndrome. He presented with intractable and recurrent convulsions, global developmental delay and microcephaly. Urine organic acid analysis using GC–MS and NMR-based urinalysis showed excessive amount of β-ureidopropionic acid and β-ureidoisobutyric acid, the two disease-specific markers for β-ureidopropionase deficiency. Genetic analysis confirmed homozygous known disease-causing mutation UPB1 NM_016327.2: c.977GNA; NP_057411.1:p.R326Q. In addition, genetic analysis for Dravet syndrome showed the presence of heterozygous disease-causing mutation SCN1A NM_001165963.1:c.4494delC; NP_001159435.1:p.F1499Lfs*2. Conclusions: The differentiation between Dravet syndrome and β-ureidopropionase deficiency is clinically challenging since both conditions share overlapping clinical features. The detection of urine β-ureidoisobutyric and β-ureidopropionic acids using NMR or GC–MS is helpful in laboratory diagnosis of β-ureidopropionase deficiency. The disease-causing mutation, c.977GNA of β-ureidopropionase deficiency, is highly prevalent in Chinese population (allele frequency = 1.7%); β-ureidopropionase deficiency screening test should be performed for any patients with unexplained neurological deficit, developmental delay or autism. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Beta-ureidopropionase deficiency is a rare inborn error of metabolism first described in 2001 using structural-based nuclear magnetic resonance (NMR) spectroscopy by Moolenaar et al. [1] In their study, the affected patient was a 22-month-old girl who presented with hypotonia and microcephaly. MRI brain showed cerebral atrophy and delayed myelination. NMR-based urinalysis persistently showed excessive amount of unknown metabolite in multiple samples. The molecular structure of this unprecedented compound was determined to be β-ureidoisobutyric acid. Together with the excessive amount of urine β-ureidopropionic acid, β-ureidopropionase (EC 3.5.1.6) was determined to be the enzymatic block in the affected patient biochemically (Fig. 1).
⁎ Corresponding author. Tel.: +852 2255 5655; fax: 852 2255 9915. E-mail address: [email protected] (C.-W. Lam).
http://dx.doi.org/10.1016/j.cca.2014.10.030 0009-8981/© 2014 Elsevier B.V. All rights reserved.
Since then, about 30 genetically confirmed cases had been reported in the literature. [2]. Recently, the molecular analysis of UPB1 gene in affected Japanese patients identified p.R326Q to be a common mutation in patients with β-ureidopropionase deficiency. Intriguingly, the same mutation was also highly prevalent in unaffected healthy Japanese subjects with an allele frequency of 0.9% [2] which was consistent with the high incidence rate of the disease (1 in 6000) in a screening of 24,000 Japanese newborns using GC-MS [3]. Thus, this apparently “rare” condition is likely to be underdiagnosed. In particular the clinical presentation of this condition is variable which could pose a further diagnostic challenge to the clinician. Indeed, some patients were asymptomatic while others could present with hypotonia, seizure, microcephaly, mental retardation, developmental delay and autism [2]. These neurological presentations were thought to be associated with intracerebral or neuronal deficiency of β-alanine, the end product of uracil after the three-step pyrimidine metabolism [4]. Beta-ureidopropionase deficiency has never been reported in Hong Kong Chinese. Here, we report a case of β-ureidopropionase deficiency
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C.-W. Lam et al. / Clinica Chimica Acta 440 (2015) 201–204
Despite anti-epileptic drug treatment and good compliance, the parents reported increase frequency of attacks. The frequency of GTC could reach 6 times per month and the seizure attack was not precipitated by any febrile illness. The dosage of topiramate was stepped up to 37.5 mg/37.5 mg/50 mg but it still failed to control the attack. Sodium valproate was further stepped up to 100 mg thrice daily since the last follow-up at 4 years old. Assessment of developmental milestone at 14 months old revealed delay of motor, cognitive and speech developments. Growth chart recorded from 14 months to 3 y showed head circumference and body weight below the 3rd percentile and body height at the 50th percentile. MRI brain was arranged when the patient was at 13-months old and showed no abnormality. 3. Materials and methods 3.1. GC–MS-based and NMR-based urinalysis
Fig. 1. Pyrimidine metabolism and the metabolic block at β-ureidopropionase.
in a Chinese patient with Dravet Syndrome associated mutation. The patient presented with recurrent and intractable convulsion since 4 months old. Molecular analysis of SCN1A confirmed possibly de novo mutation of Dravet syndrome. Urine biochemical analysis by gas chromatography–mass spectrometry (GC–MS) and 2-dimension (1H13C Heteronuclear Single Quantum Correlation, HSQC) NMR spectroscopy suggested a biochemical diagnosis of β-ureidopropionase deficiency. The dual molecular diagnoses were further confirmed by molecular analysis of UPB1 and SCN1A genes. We envisage that βureidopropionase deficiency is not an uncommon condition and should be considered in patients with autism, mental retardation and seizure. Dual molecular diagnoses should also be considered if the clinical presentations and biochemical phenotypes cannot be fully explained by a single gene disorder. 2. Case report The proband is a five-year-old Chinese boy born to nonconsanguineous parents with unremarkable birth history. He presented with recurrent afebrile convulsion since 4-month-old and electroencephalogram (EEG) revealed abnormal left temporal spike waves. No structural abnormality was identified by computed tomography (CT) of the brain. Piracetam, gabapentin and amino acid supplements were given to the patient but they all failed to control the seizure. The patient was admitted at 6-month-old for further management. The epileptic attacks were usually preceded by sudden eye staring, followed by generalized tonic–clonic convulsion (GTC) around 10 min. The parents found the frequency of the attacks increased from once weekly to several times weekly and the attacks were precipitated by febrile illnesses. Video EEG performed showed one epileptic attack after photic stimulation with associated ictal discharges. Physical examination showed normal neurological examination and developmental milestones. His head circumference was 47 cm (97th percentile), body weight was 7.1 kg (2nd–10th percentile) and body height was 61 cm (b 3rd percentile). Laboratory investigations for complete blood count, liver and renal function tests, blood random glucose, ammonia and uric acid levels were all unremarkable. There was no metabolic acidosis. He was given phenobarbitone. However, in view of the mixed seizure pattern including generalized tonic, tonic–clonic and myoclonic convulsions, sodium valproate (80 mg thrice daily) and topiramate (12.5 mg daily) were given to the patient.
Details of GC–MS based urine organic acid analysis and 1H NMR based urinalysis had been described in [5–7]. For 2D heteronuclear single-quantum correlation (HSQC), the pH of the urine sample was further adjusted to 2.0 and the NMR spectra were acquired using Bruker pulse sequence, “hsqcetgpprsisp2.2” on a BrukerAvance 600 MHz NMR spectrometer at 298 K. Spectra were referenced to the internal standard, 3-(trimethylsilyl)propionic-2,2,3,3-d4 (TSP) at 0.00 ppm. The overall analytical time of both 1H and 2D HSQC NMR experiment was about 30 min. Peak annotation of the NMR spectra was performed using Chenomx (NMR suite 7.0, Chenomx), BrukerBiofluid Reference Compound Database (BBIOREFCODE 2.0.0, Bruker) and published literature [1]. 3.2. Mutational analysis of the UPB1 and SCN1A genes Blood samples were collected from the proband and his parents after informed consent. Extraction of genomic DNA was performed using Qiagen DNA extraction kit (Qiagen). Methods for polymerase chain reaction (PCR) and sequencing are described in [8]. Primer sequences are available upon request. Sequencing results of all coding exons and the flanking intronic regions of the UPB1 and SCN1A genes were compared with the National Center for Biotechnology Information (NCBI). Confirmation of indel and sequence alignment was performed using PrimeIndel (ver 2.0) (available online — http://hpcf.cgs.hku.hk/ primeindel/indelchecker2) [9]. 4. Results Urine metabolic screening using GC–MS detected excessive pyrimidine metabolites, including marked increase of β-ureidopropionic and β-ureidoisobutyric acid, moderate increase of dihydrouracil and dihydrothymine, and mild increase of uracil and thymine. Using 1H and 2D HSQC NMR spectroscopy, excessive β-ureidoisobutyric acid and β-ureidopropionic acid were found in urine sample of the proband and their chemical identities were confirmed by their proton and carbon resonances (Fig. 2a). The excessive amount of urine β-ureidoisobutyric acid and β-ureidopropionic acid suggested the enzymatic block at β-ureidopropionase. In contrast, β-ureidoisobutyric acid and β-ureidopropionic acid were undetected in our control samples using 1H NMR spectroscopy. We had also performed 2D HSQC NMR spectroscopy using urine sample from control (Fig. 2b), both βureidoisobutyric acid and β-ureidopropionic acid were undetected in HSQC spectra after signal enhancement. Mutation analysis for UPB1 gene of the proband showed a homozygous mutation NM_016327.2: c.977GNA; NP_057411.1:p.R326Q and a heterozygous mutation NM_016327.2: c.91GNA; NP_057411.1:p.G31S. Both mutations were known to be pathogenic and cause βureidopropionase deficiency [2]. Genetic analysis of the parents revealed heterozygous c.977GNA in the mother while the father harbored
C.-W. Lam et al. / Clinica Chimica Acta 440 (2015) 201–204
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Fig. 2. HSQC spectrum showing the presence of β-ureidoisobutyric acid (square) and β-ureidopropionic acid (diamond) in urine sample from the patient (A). HSQC spectrum of urine samples from normal control (B). β-Ureidoisobutyric acid and β-ureidopropionic acid, the two metabolic signatures of beta-ureidopropionase deficiency were not detected in (B).
both heterozygous c.91GNA and c.977GNA. In addition, mutation analysis for SCN1A gene of the proband showed a heterozygous mutation NM_001165963.1:c.4494delC; NP_001159435.1:p.F1499Lfs*2 which confirmed Dravet syndrome (Supp. Fig. 1). 5. Discussion The differentiation between Dravet syndrome and β-ureidopropionase deficiency can be clinically challenging since both conditions showed overlapping clinical features, for example, microcephaly, tonic–clonic seizure, status epilepticus, refractory to anticonvulsants, developmental delay, mental retardation and cortical blindness [10,11]. Indeed, febrile convulsion had also been described in some patients with β-ureidopropionase deficiency [12]. The detection of urine β-ureidoisobutyric and β-ureidopropionic acids is crucial in laboratory
diagnosis of β-ureidopropionase deficiency. For patients with suspected Dravet syndrome, biochemical screening for β-ureidopropionase deficiency should be followed using NMR or GC–MS since the condition is not uncommon; the disease-causing mutation, c.977GNA, was prevalent in Northern Chinese and Japanese population with an allele frequency of 2.8% and 0.9% respectively [2,13]. We also determined that c.977GNA was prevalent in Hong Kong Chinese population with an allele frequency of 1.7%, which the result was the same as those reported from the 1000 Genomes Project [14]. The expected frequency for carrier and cases in Chinese population deduced from Hardy– Weinberg equilibrium will be about 3% and 0.03%. For any patients with β-ureidopropionase deficiency, clinicians and pathologists should inform the patients about the risk of 5-fluorouracil-related toxicity [15]. On the other hand, for patients with β-ureidopropionase deficiency, the differential diagnosis of Dravet syndrome should also be considered
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because carbamazepine and lamotrigine may aggregate seizure while sodium valproate, benzodiazepines and topiramate are more effective in controlling the attacks [16,17]. With the advance of fully automatic NMR spectroscopy, rapid screening of β-ureidopropionase deficiency is now within our reach and it should be considered in any patients with unexplained neurological deficit, developmental delay or autism. The routine 1H and 2D HSQC NMR analysis takes about 30 min. Usually, a 1H spectrum (15 min) is adequate for diagnostic purpose. Since the paired proton and carbon NMR assignments are unique to the chemical structure, a 2D HSQC analysis could provide structural-based evidence on the positive identification of metabolites. This information is particularly useful for revealing the identity of unknown metabolites in novel IEM as described by Moolenaar et al.[1]. The reason of the variable clinical manifestation in patients with β-ureidopropionase deficiency is not fully understood. Very recently, Rubinstein et al. demonstrated that the excitability of inhibitory neurons of Dravet Syndrome mice in 129/SvJ genetic background was less impaired than that of mice in C57BL/6 genetic background. Thus, different genetic backgrounds may affect the phenotype of Scn1a mutant mice [18]. The phenotype of UPB1 knock-out mice has not been published, it is possible that additional genetic or environmental factors may interact with the UPB1 gene in the disease manifestation, for example 5-fluorouracil-related toxicity in βureidopropionase deficiency [15]. Nevertheless, dual molecular diagnoses are not uncommon (occurrence rate = 1.6%) [19] which may explain the phenotype observed in the proband. Molecular genetic tests and urine metabolic analysis could help resolved this diagnostic challenge with impacts on clinical management and genetic counseling to the family. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cca.2014.10.030.
Acknowledgments This work was supported by the Research Fund for the Control of Infectious Diseases (RFCID) from the Food and Health Bureau, the Government of Hong Kong SAR.
References [1] Moolenaar SH, Gohlich-Ratmann G, Engelke UF, et al. Beta-ureidopropionase deficiency: a novel inborn error of metabolism discovered using NMR spectroscopy on urine. Magn Reson Med 2001;46:1014–7. [2] Nakajima Y, Meijer J, Dobritzsch D, et al. Clinical, biochemical and molecular analysis of 13 Japanese patients with beta-ureidopropionase deficiency demonstrates high prevalence of the c.977GNA (p.R326Q) mutation. J Inherit Metab Dis 2014;37: 801–12. [3] Kuhara T, Ohse M, Inoue Y, et al. Five cases of beta-ureidopropionase deficiency detected by GC/MS analysis of urine metabolome. J Mass Spectrom 2009;44:214–21. [4] Assmann B, Gohlich G, Baethmann M, et al. Clinical findings and a therapeutic trial in the first patient with beta-ureidopropionase deficiency. Neuropediatrics 2006;37: 20–5. [5] Lee HC, Lai CK, Yau KC, et al. Non-invasive urinary screening for aromatic L-amino acid decarboxylase deficiency in high-prevalence areas: a pilot study. Clin Chim Acta 2012;413:126–30. [6] Lam CW, Law CY, To KK, et al. NMR-based metabolomic urinalysis: a rapid screening test for urinary tract infection. Clin Chim Acta 2014;436C:217–23. [7] Law CY, Lam CW, Ching CK, et al. NMR-based urinalysis for beta-ketothiolase deficiency. Clin Chim Acta 2014;438C:222–5. [8] Siu WK, Law CY, Lam CW, et al. Novel nonsense CDC73 mutations in Chinese patients with parathyroid tumors. Fam Cancer 2011;10:695–9. [9] Lam CW. PrimeIndel: four-prime-number genetic code for indel decryption and sequence read alignment. Clin Chim Acta 2014;436:1–4. [10] Online Mendelian Inheritance in Man, OMIM®. Johns Hopkins University, Baltimore, MD. MIM Number: #607208:04/22/2014. World Wide Web URL: http://omim.org/. [Assessed on October 2014]. [11] Online Mendelian Inheritance in Man, OMIM®. Johns Hopkins University, Baltimore, MD. MIM Number: #613161:01/31/2011. World Wide Web URL: http://omim.org/. [Assessed on October 2014]. [12] Assmann BE, Van Kuilenburg AB, Distelmaier F, et al. Beta-ureidopropionase deficiency presenting with febrile status epilepticus. Epilepsia 2006;47:215–7. [13] Shu J, Lv X, Jiang S, et al. Genetic analysis of the UPB1 gene in two new Chinese families with beta-ureidopropionase deficiency and the carrier frequency of the mutation c.977GNA in Northern China. Childs Nerv Syst 2014;30:2109–14. [14] Abecasis GR, Auton A, Brooks LD, et al. An integrated map of genetic variation from 1,092 human genomes. Nature 2012;491:56–65. [15] Fidlerova J, Kleiblova P, Kormunda S, Novotnyand J, Kleibl Z. Contribution of the beta-ureidopropionase (UPB1) gene alterations to the development of fluoropyrimidine-related toxicity. Pharmacol Rep 2012;64:1234–42. [16] Brunklaus A, Ellis R, Reavey E, Forbes GH, Zuberi SM. Prognostic, clinical and demographic features in SCN1A mutation-positive Dravet syndrome. Brain 2012; 135:2329–36. [17] Guerrini R, Dravet C, Genton P, et al. Lamotrigine and seizure aggravation in severe myoclonic epilepsy. Epilepsia 1998;39:508–12. [18] Rubinstein M, Westenbroek RE, Yu FH, Jones CJ, Scheuer T, et al. Genetic background modulates impaired excitability of inhibitory neurons in a mouse model of Dravet syndrome. Neurobiol Dis 2014;73C:106–17. [19] Yang Y, Muzny DM, Reid JG, et al. Clinical whole-exome sequencing for the diagnosis of Mendelian disorders. N Engl J Med 2013;369:1502–11.
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Case report
NMR-based urinalysis for rapid diagnosis of β-ureidopropionase deficiency in a patient with Dravet syndrome Ching-Wan Lam a,⁎, Chun-Yiu Law a, Ka-Fei Leung a, Chi-Kong Lai b, Sammy Pak-lam Chen b, Bosco Chan c, Kwok-yin Chan c, Yuet-ping Yuen b, Chloe Miu Mak b, Albert Yan-wo Chan b a b c
Department of Pathology, The University of Hong Kong, Hong Kong, China Department of Pathology, Princess Margaret Hospital, Hong Kong, China Department of Paediatrics and Adolescent Medicine, Princess Margaret Hospital, Hong Kong, China
a r t i c l e
i n f o
Article history: Received 13 October 2014 Received in revised form 17 October 2014 Accepted 21 October 2014 Available online 25 October 2014 Keywords: NMR spectroscopy β-Ureidopropionase deficiency Dravet syndrome Dual molecular diagnoses
a b s t r a c t Background: Beta-ureidopropionase deficiency is a rare inborn error of metabolism (IEM) affecting pyrimidine metabolism. To-date, about 30 genetically confirmed cases had been reported. The clinical phenotypes of this condition are variable; some patients were asymptomatic while some may present with developmental delay or autistic features. In severe cases, patients may present with profound neurological deficit including hypotonia, seizures and mental retardation. Using NMR-based urinalysis, this condition can be rapidly diagnosed within 15 min. Case: An 11-month-old Chinese boy had dual molecular diagnoses, β-ureidopropionase deficiency and Dravet syndrome. He presented with intractable and recurrent convulsions, global developmental delay and microcephaly. Urine organic acid analysis using GC–MS and NMR-based urinalysis showed excessive amount of β-ureidopropionic acid and β-ureidoisobutyric acid, the two disease-specific markers for β-ureidopropionase deficiency. Genetic analysis confirmed homozygous known disease-causing mutation UPB1 NM_016327.2: c.977GNA; NP_057411.1:p.R326Q. In addition, genetic analysis for Dravet syndrome showed the presence of heterozygous disease-causing mutation SCN1A NM_001165963.1:c.4494delC; NP_001159435.1:p.F1499Lfs*2. Conclusions: The differentiation between Dravet syndrome and β-ureidopropionase deficiency is clinically challenging since both conditions share overlapping clinical features. The detection of urine β-ureidoisobutyric and β-ureidopropionic acids using NMR or GC–MS is helpful in laboratory diagnosis of β-ureidopropionase deficiency. The disease-causing mutation, c.977GNA of β-ureidopropionase deficiency, is highly prevalent in Chinese population (allele frequency = 1.7%); β-ureidopropionase deficiency screening test should be performed for any patients with unexplained neurological deficit, developmental delay or autism. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Beta-ureidopropionase deficiency is a rare inborn error of metabolism first described in 2001 using structural-based nuclear magnetic resonance (NMR) spectroscopy by Moolenaar et al. [1] In their study, the affected patient was a 22-month-old girl who presented with hypotonia and microcephaly. MRI brain showed cerebral atrophy and delayed myelination. NMR-based urinalysis persistently showed excessive amount of unknown metabolite in multiple samples. The molecular structure of this unprecedented compound was determined to be β-ureidoisobutyric acid. Together with the excessive amount of urine β-ureidopropionic acid, β-ureidopropionase (EC 3.5.1.6) was determined to be the enzymatic block in the affected patient biochemically (Fig. 1).
⁎ Corresponding author. Tel.: +852 2255 5655; fax: 852 2255 9915. E-mail address: [email protected] (C.-W. Lam).
http://dx.doi.org/10.1016/j.cca.2014.10.030 0009-8981/© 2014 Elsevier B.V. All rights reserved.
Since then, about 30 genetically confirmed cases had been reported in the literature. [2]. Recently, the molecular analysis of UPB1 gene in affected Japanese patients identified p.R326Q to be a common mutation in patients with β-ureidopropionase deficiency. Intriguingly, the same mutation was also highly prevalent in unaffected healthy Japanese subjects with an allele frequency of 0.9% [2] which was consistent with the high incidence rate of the disease (1 in 6000) in a screening of 24,000 Japanese newborns using GC-MS [3]. Thus, this apparently “rare” condition is likely to be underdiagnosed. In particular the clinical presentation of this condition is variable which could pose a further diagnostic challenge to the clinician. Indeed, some patients were asymptomatic while others could present with hypotonia, seizure, microcephaly, mental retardation, developmental delay and autism [2]. These neurological presentations were thought to be associated with intracerebral or neuronal deficiency of β-alanine, the end product of uracil after the three-step pyrimidine metabolism [4]. Beta-ureidopropionase deficiency has never been reported in Hong Kong Chinese. Here, we report a case of β-ureidopropionase deficiency
202
C.-W. Lam et al. / Clinica Chimica Acta 440 (2015) 201–204
Despite anti-epileptic drug treatment and good compliance, the parents reported increase frequency of attacks. The frequency of GTC could reach 6 times per month and the seizure attack was not precipitated by any febrile illness. The dosage of topiramate was stepped up to 37.5 mg/37.5 mg/50 mg but it still failed to control the attack. Sodium valproate was further stepped up to 100 mg thrice daily since the last follow-up at 4 years old. Assessment of developmental milestone at 14 months old revealed delay of motor, cognitive and speech developments. Growth chart recorded from 14 months to 3 y showed head circumference and body weight below the 3rd percentile and body height at the 50th percentile. MRI brain was arranged when the patient was at 13-months old and showed no abnormality. 3. Materials and methods 3.1. GC–MS-based and NMR-based urinalysis
Fig. 1. Pyrimidine metabolism and the metabolic block at β-ureidopropionase.
in a Chinese patient with Dravet Syndrome associated mutation. The patient presented with recurrent and intractable convulsion since 4 months old. Molecular analysis of SCN1A confirmed possibly de novo mutation of Dravet syndrome. Urine biochemical analysis by gas chromatography–mass spectrometry (GC–MS) and 2-dimension (1H13C Heteronuclear Single Quantum Correlation, HSQC) NMR spectroscopy suggested a biochemical diagnosis of β-ureidopropionase deficiency. The dual molecular diagnoses were further confirmed by molecular analysis of UPB1 and SCN1A genes. We envisage that βureidopropionase deficiency is not an uncommon condition and should be considered in patients with autism, mental retardation and seizure. Dual molecular diagnoses should also be considered if the clinical presentations and biochemical phenotypes cannot be fully explained by a single gene disorder. 2. Case report The proband is a five-year-old Chinese boy born to nonconsanguineous parents with unremarkable birth history. He presented with recurrent afebrile convulsion since 4-month-old and electroencephalogram (EEG) revealed abnormal left temporal spike waves. No structural abnormality was identified by computed tomography (CT) of the brain. Piracetam, gabapentin and amino acid supplements were given to the patient but they all failed to control the seizure. The patient was admitted at 6-month-old for further management. The epileptic attacks were usually preceded by sudden eye staring, followed by generalized tonic–clonic convulsion (GTC) around 10 min. The parents found the frequency of the attacks increased from once weekly to several times weekly and the attacks were precipitated by febrile illnesses. Video EEG performed showed one epileptic attack after photic stimulation with associated ictal discharges. Physical examination showed normal neurological examination and developmental milestones. His head circumference was 47 cm (97th percentile), body weight was 7.1 kg (2nd–10th percentile) and body height was 61 cm (b 3rd percentile). Laboratory investigations for complete blood count, liver and renal function tests, blood random glucose, ammonia and uric acid levels were all unremarkable. There was no metabolic acidosis. He was given phenobarbitone. However, in view of the mixed seizure pattern including generalized tonic, tonic–clonic and myoclonic convulsions, sodium valproate (80 mg thrice daily) and topiramate (12.5 mg daily) were given to the patient.
Details of GC–MS based urine organic acid analysis and 1H NMR based urinalysis had been described in [5–7]. For 2D heteronuclear single-quantum correlation (HSQC), the pH of the urine sample was further adjusted to 2.0 and the NMR spectra were acquired using Bruker pulse sequence, “hsqcetgpprsisp2.2” on a BrukerAvance 600 MHz NMR spectrometer at 298 K. Spectra were referenced to the internal standard, 3-(trimethylsilyl)propionic-2,2,3,3-d4 (TSP) at 0.00 ppm. The overall analytical time of both 1H and 2D HSQC NMR experiment was about 30 min. Peak annotation of the NMR spectra was performed using Chenomx (NMR suite 7.0, Chenomx), BrukerBiofluid Reference Compound Database (BBIOREFCODE 2.0.0, Bruker) and published literature [1]. 3.2. Mutational analysis of the UPB1 and SCN1A genes Blood samples were collected from the proband and his parents after informed consent. Extraction of genomic DNA was performed using Qiagen DNA extraction kit (Qiagen). Methods for polymerase chain reaction (PCR) and sequencing are described in [8]. Primer sequences are available upon request. Sequencing results of all coding exons and the flanking intronic regions of the UPB1 and SCN1A genes were compared with the National Center for Biotechnology Information (NCBI). Confirmation of indel and sequence alignment was performed using PrimeIndel (ver 2.0) (available online — http://hpcf.cgs.hku.hk/ primeindel/indelchecker2) [9]. 4. Results Urine metabolic screening using GC–MS detected excessive pyrimidine metabolites, including marked increase of β-ureidopropionic and β-ureidoisobutyric acid, moderate increase of dihydrouracil and dihydrothymine, and mild increase of uracil and thymine. Using 1H and 2D HSQC NMR spectroscopy, excessive β-ureidoisobutyric acid and β-ureidopropionic acid were found in urine sample of the proband and their chemical identities were confirmed by their proton and carbon resonances (Fig. 2a). The excessive amount of urine β-ureidoisobutyric acid and β-ureidopropionic acid suggested the enzymatic block at β-ureidopropionase. In contrast, β-ureidoisobutyric acid and β-ureidopropionic acid were undetected in our control samples using 1H NMR spectroscopy. We had also performed 2D HSQC NMR spectroscopy using urine sample from control (Fig. 2b), both βureidoisobutyric acid and β-ureidopropionic acid were undetected in HSQC spectra after signal enhancement. Mutation analysis for UPB1 gene of the proband showed a homozygous mutation NM_016327.2: c.977GNA; NP_057411.1:p.R326Q and a heterozygous mutation NM_016327.2: c.91GNA; NP_057411.1:p.G31S. Both mutations were known to be pathogenic and cause βureidopropionase deficiency [2]. Genetic analysis of the parents revealed heterozygous c.977GNA in the mother while the father harbored
C.-W. Lam et al. / Clinica Chimica Acta 440 (2015) 201–204
203
Fig. 2. HSQC spectrum showing the presence of β-ureidoisobutyric acid (square) and β-ureidopropionic acid (diamond) in urine sample from the patient (A). HSQC spectrum of urine samples from normal control (B). β-Ureidoisobutyric acid and β-ureidopropionic acid, the two metabolic signatures of beta-ureidopropionase deficiency were not detected in (B).
both heterozygous c.91GNA and c.977GNA. In addition, mutation analysis for SCN1A gene of the proband showed a heterozygous mutation NM_001165963.1:c.4494delC; NP_001159435.1:p.F1499Lfs*2 which confirmed Dravet syndrome (Supp. Fig. 1). 5. Discussion The differentiation between Dravet syndrome and β-ureidopropionase deficiency can be clinically challenging since both conditions showed overlapping clinical features, for example, microcephaly, tonic–clonic seizure, status epilepticus, refractory to anticonvulsants, developmental delay, mental retardation and cortical blindness [10,11]. Indeed, febrile convulsion had also been described in some patients with β-ureidopropionase deficiency [12]. The detection of urine β-ureidoisobutyric and β-ureidopropionic acids is crucial in laboratory
diagnosis of β-ureidopropionase deficiency. For patients with suspected Dravet syndrome, biochemical screening for β-ureidopropionase deficiency should be followed using NMR or GC–MS since the condition is not uncommon; the disease-causing mutation, c.977GNA, was prevalent in Northern Chinese and Japanese population with an allele frequency of 2.8% and 0.9% respectively [2,13]. We also determined that c.977GNA was prevalent in Hong Kong Chinese population with an allele frequency of 1.7%, which the result was the same as those reported from the 1000 Genomes Project [14]. The expected frequency for carrier and cases in Chinese population deduced from Hardy– Weinberg equilibrium will be about 3% and 0.03%. For any patients with β-ureidopropionase deficiency, clinicians and pathologists should inform the patients about the risk of 5-fluorouracil-related toxicity [15]. On the other hand, for patients with β-ureidopropionase deficiency, the differential diagnosis of Dravet syndrome should also be considered
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because carbamazepine and lamotrigine may aggregate seizure while sodium valproate, benzodiazepines and topiramate are more effective in controlling the attacks [16,17]. With the advance of fully automatic NMR spectroscopy, rapid screening of β-ureidopropionase deficiency is now within our reach and it should be considered in any patients with unexplained neurological deficit, developmental delay or autism. The routine 1H and 2D HSQC NMR analysis takes about 30 min. Usually, a 1H spectrum (15 min) is adequate for diagnostic purpose. Since the paired proton and carbon NMR assignments are unique to the chemical structure, a 2D HSQC analysis could provide structural-based evidence on the positive identification of metabolites. This information is particularly useful for revealing the identity of unknown metabolites in novel IEM as described by Moolenaar et al.[1]. The reason of the variable clinical manifestation in patients with β-ureidopropionase deficiency is not fully understood. Very recently, Rubinstein et al. demonstrated that the excitability of inhibitory neurons of Dravet Syndrome mice in 129/SvJ genetic background was less impaired than that of mice in C57BL/6 genetic background. Thus, different genetic backgrounds may affect the phenotype of Scn1a mutant mice [18]. The phenotype of UPB1 knock-out mice has not been published, it is possible that additional genetic or environmental factors may interact with the UPB1 gene in the disease manifestation, for example 5-fluorouracil-related toxicity in βureidopropionase deficiency [15]. Nevertheless, dual molecular diagnoses are not uncommon (occurrence rate = 1.6%) [19] which may explain the phenotype observed in the proband. Molecular genetic tests and urine metabolic analysis could help resolved this diagnostic challenge with impacts on clinical management and genetic counseling to the family. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cca.2014.10.030.
Acknowledgments This work was supported by the Research Fund for the Control of Infectious Diseases (RFCID) from the Food and Health Bureau, the Government of Hong Kong SAR.
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