Eleven Novel Mutations of the BCKDHA, BCKDHB and DBT Genes Associated with Maple Syrup Urine Disease in the Chinese Population: Report on Eight Cases

European Journal of Medical Genetics 58 (2015) 617e623

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European Journal of Medical Genetics

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Clinical research Eleven novel mutations of the BCKDHA, BCKDHB and DBT genes associated with maple syrup urine disease in the Chinese population: Report on eight cases

Xiyuan Li a, Yuan Ding a, Yupeng Liu a, Yanyan Ma a, Jinqing Song a, Qiao Wang a, * Mengqiu Li b, Yaping Qin b, Yanling Yang a, a Department of Pediatrics, Peking University First Hospital, Beijing 100034, China b Similan Clinic, Beijing 100070, China article info abstract

Article history: Maple syrup urine disease (MSUD) is a rare autosomal recessive disorder that affects the degradation of Received 17 March 2015 branched chain amino acids (BCAAs). Only a few cases of MSUD have been documented in Mainland Received in revised form China, and prenatal diagnosis has not been performed so far. In this report, 8 patients (4 girls and 4 boys) 30 September 2015 with MSUD from 8 unrelated Chinese families were diagnosed at the age of 9 days to 1 year and 8 Accepted 3 October 2015 months. The diagnosis was confirmed by serum BCAAs and genetic analyses. Among the 8 patients, only Available online 8 October 2015 one was detected by newborn screening. The remaining 7 patients were admitted because of neuro- logical disorders and underwent selective screening. Significantly elevated BCAAs were observed in 7 Keywords: Maple syrup urine disease patients. One patient was diagnosed by post-mortem study. 12 mutations were found in the BCKDHA, > > > Branched chain amino acids BCKDHB and DBT genes. 11 of these mutations were novel: c.178G T, c.491T C, c.740A G, BCKDHA gene c.1214_1219dupCCAACC and IVS6 þ 1delG in BCKDHA; c.482T > G, c.508C > T, c.767A > G, c.768C > G and BCKDHB gene IVS4,2A > CinBCKDHB; and c.1A > GinDBT. Only one mutation, c.659C > T in the BCKDHA gene, had DBT gene been previously reported. 7 patients were treated by dietary intervention and symptomatic therapy. 6 of Prenatal diagnosis them showed clinical improvement. The mother of one patient who died from MSUD underwent amniocentesis during her second pregnancy. The BCAAs level in her amniotic fluid was normal. Only one heterozygous mutation, IVS4,2A > C in the BCKDHB gene, was detected in the cultured amniocytes. The results revealed that the fetus was not affected by MSUD. Normal development and the blood BCAAs profile confirmed the prenatal diagnosis after birth. Thus, we identified eleven novel mutations associated with MSUD in the Chinese population. Prenatal diagnosis of MSUD was successfully performed on one fetus by genetic analysis of the cultured amniocytes. © 2015 Elsevier Masson SAS. All rights reserved.

1. Introduction DiGeorge, 1981). The disease was first reported by Menkes JH in 1954 (Menkes et al., 1954), and hundreds of cases have been Maple syrup urine disease (MSUD, OMIM #248600) is a rare detected to date. autosomal recessive disorder that affects degradation of the BCKDC defects result in a remarkable increase in the amount of branched chain amino acids (BCAAs) leucine, valine, and isoleu- BCAAs and alloisoleucine, and their a-keto acids (a-ketoisocap- cine; it is caused by defects in the branched-chain a-keto acid de- roate, a-keto-b-methylvalerate and a-ketoisovalerate) in the hydrogenase complex (BCKDC). The worldwide incidence of MSUD plasma, urine and cerebrospinal fluid (Strauss et al., 1993). Accu- is estimated to be 1 in 185,000 (Chuang et al., 2006; Harper et al., mulation of these BCAAs and their ɑ-keto acids is toxic to the 1990), but it is as high as 1 in 176 live births in certain consan- central nervous system. BCAAs in the brain compete with other guineous ethnic Mennonite groups in Pennsylvania (Marshall and amino acids for binding to the neutral amino acid transporter, which is responsible for the uptake of large essential neutral amino acids in the brain; this consequently results in a deficiency of large neutral amino acids in the brain (Killian and Chikhale, 2001). En- * Corresponding author. ergy deficiency as a result of disruption of Kreb's cycle is also E-mail addresses: [email protected], [email protected] (Y. Yang). http://dx.doi.org/10.1016/j.ejmg.2015.10.002 1769-7212/© 2015 Elsevier Masson SAS. All rights reserved. 618 X. Li et al. / European Journal of Medical Genetics 58 (2015) 617e623 responsible for brain injury in MSUD patients (Zinnanti et al., conducted. 2009). In addition, during certain non-specific illnesses that result Urinary organic acids were analyzed by gas chromatography- in metabolic crises, such as fever, infection and diarrhea, metabolic mass spectrometry (GCMS) using a GCMS-QP2010 analyzer (Shi- decomposition occurs and worsens the prognosis of these patients madzu, Japan) and the Inborn Errors of Metabolism Screening (Strauss et al., 1993). Furthermore, MSUD leads to a series of System software for the differential diagnosis of organic acidurias neuropathological defects, such as depletion of glutamate, gluta- (Fu et al., 2000; Kimura et al., 1999). mine and g-aminobutyric acid; disturbance in neurotransmitter Blood amino acids, free carnitine, and acylcarnitines were balance; increased apoptosis and oxidative stress (Dodd et al., 1992; analyzed by liquid chromatography-tandem mass spectrometry Huang et al., 1996; Jouvet et al., 2000). (LC-MS/MS) (Applied Biosystems, USA) using an Applied Bio- Based on the disease phenotype, MSUD is classified into 4 types: systems API 3200 analyzer and the ChemoView software for the classic, intermediate, intermittent and thiamine-responsive. The differential diagnosis of aminoacidopathies, fatty acid metabolic majority of patients (<75%) exhibit the classic form, which is also disorders, and other organic acidurias (Zytkovicz et al., 2001). the most severe form. Patients with the classic form of MSUD BCAAs in the serum and amniotic fluid were analyzed (Similan usually present with ketonuria, irritability, and poor feeding at the Clinic, Beijing, China) using GCMS, with GCMS-QP2010 PLUS (Shi- age of 2e3 days, and worsening encephalopathy, opisthotonus, madzu, Japan). intermittent apnea, and stereotyped movements at the age of 4e5 days. If left untreated, coma and even central respiratory failure can 2.3. BCKDHA, BCKDHB and DBT gene analysis occur at the age of 7e10 days (Strauss et al., 1993). The intermediate type is characterized by poor growth, poor feeding and develop- The informed consents of the parents of the patients were ob- mental delays; moreover, encephalopathy can be observed during tained for the genetic analysis. Genomic DNA was extracted from certain episodes. Patients with intermittent MSUD usually exhibit the peripheral blood lymphocytes of the patients and their parents normal development with episodes of decomposition. Patients using the TIANamp Blood DNA Kit (Tiangen Biotech, China). The with thiamine-responsive MSUD show similar manifestations to exons and flanking intronic regions of the BCKDHA, BCKDHB and patients with intermediate MSUD; however, the former present DBT genes were amplified using PCR and sequenced. The results with leucine tolerance, and their biochemical profiles usually were compared with the reference sequences of BCKDHA improve after thiamine therapy (Menkes et al., 1954; Morton et al., (NM_000709), BCKDHB (NM_183050), DLD (NM_000108) and DBT 2012; Strauss et al., 1993). (NM_001918) deposited in UCSC genome (http://genome.ucsc.edu/ The 4 subunits of BCKDC, E1a,E1b, E2, and E3, are encoded by ). Sequencing data were compared with an integrated set of vari- the BCKDHA, BCKDHB, DBT and DLD genes, respectively (Strauss ants (http://www.hgmd.cf.ac.uk), genotypes, and haplotypes from et al., 1993). To date, 48 mutations of the BCKDHA gene, 87 muta- the 1000 Genomes Project (www.1000genomes.org) in order to tions of the BCKDHB gene, 18 mutations of the DLD gene and 54 identify mutations. mutations of the DBT gene have been reported (http://www.hgmd. cf.ac.uk/ac/index.php). Founder mutations have been reported in 2.4. Prediction of the effects of mutations of BCKDHA, BCKDHB and certain populations of the world, but founder mutations have not DBT and conservation analysis been reported in the Chinese population to date. Only a few cases of MSUD have been documented in mainland Multiple sequence alignments were performed to verify the China (Wang et al., 2015; Yang et al., 2012; You et al., 2014). Here, degree of conservation. PolyPhen-2 and the Mutationtaster pro- we describe 8 patients with MSUD from 8 unrelated Chinese fam- gram were used to predict the impact of missense alterations on ilies. 11 novel mutations were identified in these patients. Further, protein function (http://genetics.bwh.harvard.edu/pph/, www. MSUD was prenatally diagnosed in one fetus. mutationtaster.org/). Multiple sequence alignments were ob- tained using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). 2. Patients and methods 2.5. Amniocentesis for prenatal diagnosis 2.1. Patients At the gestational age of 21 weeks, 20 ml amniotic fluid was 8 patients (4 girls and 4 boys) from 8 unrelated Chinese families collected from the mother of Patient 5 at the Department of Ob- were diagnosed with MSUD at the Department of Pediatrics, Peking stetrics and Gynecology, Peking University First Hospital. The am- University First Hospital, between January 2011 and December niotic fluid samples were centrifuged at 3000 rpm for 5 min at 4 C. 2014. The clinical onset of the symptoms occurred between 2 days The cell-free amniotic fluid was then separated for the metabolites and 18 months of age. They were admitted with indications of assays. The amniocytes were cultured using standard methods for hypoxic ischemic encephalopathy, neonatal septicemia, and psy- genetic analysis (Hamerton, 1982). chomotor retardation (Table 1). All the parents of the patients were healthy and non- 3. Results consanguineous. This study was approved by the Institutional Re- view Board (IRB) of the hospital and was conducted in accordance 3.1. Clinical data and laboratory examinations with the Declaration of Helsinki. The mother of Patient 5 subsequently visited us at 20 weeks of Clinical data and the results of the laboratory examinations are pregnancy, seeking genetic counseling and prenatal screening for presented in Table 1. 8 patients were born at term. Only one patient MSUD. (Patient 7) was found by newborn screening. The other 7 patients were diagnosed by selective screening. Their symptoms were 2.2. Routine tests and metabolic studies observed between the ages of 2 days and 18 months. 6 patients had very early onset, between the 2nd and 13th day of life. Patient 5 was Routine laboratory tests for blood and urine, liver and renal diagnosed by a post-mortem investigation. Patient 6 had late-onset function, serum electrolytes, glucose, ammonia, ketones, creatine MSUD at 18 months of age with movement disorder. 6 patients had kinase, creatine kinase isoenzymes and blood gas analysis were feeding difficulties. 4 had seizures. 4 had hypertonia. 7 exhibited X. Li et al. / European Journal of Medical Genetics 58 (2015) 617e623 619

Table 1 Clinical and laboratory features of eight patients with MSUD before treatment.

Patients 12345678Normal range

Gender M FFFMMFM Age of onset 3 d 4 d 12 d 5 d 2 d 1 y 6 mo 1 y 4 mo 13 d Age of diagnosis 17 d 1 mo 9 mo 5 mo 21 d 1 y 8 mo 9 d 16 d Present age 2 y 8 mo 4 y 3 mo 2 y 3 mo 3 y 11 mo Died 2 y 6 mo 2 y 5 mo 2 y 8 mo Symptoms and signs Vomiting eeþþee e e Feeding difﬁculties þþþþþeeþ Seizures þ e þþþeee Hypermyotonia þþe þþeee Maple syrup odor þþþþþþe þ Unconsciousness þ eeþþeee Psychomotor þþþþþþee retardation Positive family eeeeeeee history Cranial MRI/CT þþþþþþþþ Blood ammonia (mM) 75 95 205 129 131 45 34 96 <54 Hemoglobin (g/L) 141 117 118 59 89 124 137 98 120e160 Eleveated ɑ-keto þþþþþþþþ acids in urine Serum branched chain amino acids Leucin (mM) 175.85 745.01 891.90 1155.27 N/A 99.9 621.72 1047.24 4.20 e367.00 Valine (mM) 532.83 526.40 502.50 848.19 N/A 471.81 594.15 466.32 117.60 e495.70 Isoleucine (mM) 384.90 181.06 165.99 374.34 N/A 167.13 225.21 261.09 26.90 e146.00 Alloisoleucine (mM) 213.09 69.45 90.12 128.73 N/A 44.88 76.62 127.05 0.00e7.70 Subtypes Classical Classical Classical Classical classIcal Intermediate Unknown Classical Outcome Psychomotor Psychomotor Psychomotor Psychomotor Died Psychomotor Normal Normal retardation retardation retardation retardation retardation development development

Notes: M ¼ male, F ¼ female, y ¼ years, mo ¼ month, d ¼ day. the characteristic maple syrup odor of the urine. 7 patients had 3.1.3. Case 3 hyperammonemia, and 5 had anemia. Markedly elevated urine a- This patient was a girl who was born by vaginal delivery. Her keto acids were found in all the patients. With the exception of elder brother was healthy. She exhibited feeding difficulties and Patient 5, who was never able to be tested, the remaining 7 patients vomiting at 12 days of age. Severe psychomotor retardation was had significantly elevated BCAAs in the serum (Table 1). found at the age of 3 months. Cerebral MRI showed bilateral damage to the globus pallidus, basal ganglia, capsula interna and capsula externa. After treatment, clinical improvement was 3.1.1. Case 1 observed. Currently, she is 4 years old and has moderate psycho- Case 1 was a boy. He was born via vaginal delivery. He exhibited motor retardation. seizures on the third day of life. Hypermyotonia and feeding difficulties occurred at 5 days. When he was admitted at 15 days of age, 3.1.4. Case 4 he was unconscious, and had severe acidosis, hypoglycemia, This patient was a girl who was born by vaginal delivery. Her hyperammonemia and anemia. Cranial magnetic resonance imag- elder brother was healthy. This patient presented with lethargy and ing (MRI) revealed abnormal signals in the left and right basal feeding difficulty when she was 5 days old. She was admitted ganglia and globus pallidus. After MSUD was diagnosed, this pa- because of unconsciousness at the age of 5 months, and she was tient was treated with L-carnitine; vitamin B1, B2, and B12; L- diagnosed with MSUD accompanied by metabolic acidosis, liver arginine; and glucose infusion. Feeding with a leucine-, valine- and damage and hypermyotonia. Cerebral MRI showed abnormal sig- isoleucine-free formula was initiated. After treatment, the seizures nals in the left and right globus pallidus. Clinical improvement as attacks have become less frequent. Currently, he is 2 years and 8 well as improvement in the laboratory test results was observed months old and has moderate psychomotor retardation. Further, after treatment. She is now 3 years and 11 months old with severe his blood BCAAs and urine organic acids have decreased psychomotor retardation. She is still unable to walk or speak. significantly.

3.1.5. Case 5 3.1.2. Case 2 This boy was the first child of his parents. He was uncon- This patient was a girl who was delivered via Caesarean section. sciousness, and had feeding difficulties, seizures and hyper- Poor feeding and vomiting were observed at the age of 4 days. She myotonia at the age of 2 days. Cerebral MRI revealed abnormal was admitted at 1 month of age because of poor response to light signals in bilateral basal ganglia and globus pallidus. He died at the and sound. MSUD with hypermyotonia and acidosis was diagnosed. age of 21 days. The clinical diagnosis was central respiratory failure. Cerebral MRI revealed abnormal signals in the left and right frontal MSUD was diagnosed by post-mortem analysis. lobes and globus pallidus. Her response was significantly improved after treatment with vitamin B1 and a special diet. She is 4 years 3.1.6. Case 6 and 3 months old now, with mild psychomotor retardation. This vaginal delivered boy was the third child in the family. This 620 X. Li et al. / European Journal of Medical Genetics 58 (2015) 617e623 patient presented with mild developmental retardation. When he mutationsdc.482T > G, c.508C > T, c.767A > G, c.768C > Gand was 18 months old, dystonia was noticed. Cerebral MRI showed IVS4, 2A > Cdwere identified in the BCKDHB gene in patients 5e7. 1 bilateral damage to the globus pallidus, basal ganglia, cerebellar novel mutation, c.1A > G, was detected in the DBT gene in patient 8. dentate nucleus, brainstem, corpora quadrigemina, frontal lobe and The mutations that were found in the patients were detected in insular cortex. After treatment, his movement improved. He is 2 their parents too. None of these novel mutations were detected in years and 6 months old and has mild psychomotor retardation. the 200 normal controls or found in the 1000 Genomes Project (www.1000genomes.org) database for the Chinese population. 3.1.7. Case 7 Sequence alignment of the BCKDC E1a subunit revealed that 3 The girl was delivered by vaginal and detected by newborn amino acidsdL164, A220, and H247dwere highly conserved in screening. Dietary treatment was initiated at 9 days of life. How- humans, Pan troglodytes, Mus musculus, Trubripes, Danio rerio, ever, abnormal signals in thalamencephalon and cerebral ganglia Drosophila melanogaster and Xenopus tropicalis. E60 was conserved were detected via cerebral MRI. She is 2 years and 5 months old in mammals. However, P405 was not conserved (Table 3). Y256 in now and shows normal development. the E1b subunit and M1 in the E2 subunit were highly conserved in humans, Pan troglodytes, Macaca mulatta, M. musculus, Gallus gallus and X. tropicalis. L164 and R170 in the E1b subunit were partly 3.1.8. Case 8 conserved (Table 4). Except for c.1A > G in the DBT gene, the other The boy was born by cesarean delivery. He developed feeding missense mutations were predicted to be “probably damaging” and difficulties and lethargy when he was 13 days old and was admitted “disease causing” by PolyPhen-2.0 and Mutationtaster on suspicion of neonatal septicemia. Cerebral MRI revealed leuko- independently. dystrophy and corpus callosum dysplasia. Dietary treatment was initiated at 16 days of life. He is now 30 months old with normal psychomotor development. 3.4. Prenatal diagnosis

3.2. Laboratory examinations The mother of Patient 5 underwent amniocentesis at 31 years of age. The BCAAs and a-keto acids in the amniotic fluid were normal. Except for Patient 5, the remaining 7 patients exhibited signif- Only 1 heterozygous mutation, IVS4,2A > C in the BCKDHB gene, icantly elevated blood BCAAs and urine a-keto acids (Table 1). Their was found in the amniocytes. The results of the biochemical anal- serum alloisoleucine had increased to 69.45e213.09 mM (normal ysis and mutation study indicated that the fetus was not affected by range, 0.00e7.70 mM) before treatment. After dietary intervention, MSUD. The boy was born by vaginal delivery. Normal serum amino the blood BCAAs and urine a-keto acids decreased. acid levels and urine organic acid profile confirmed the prenatal diagnosis. Currently, the boy is 18 months old and shows normal psychomotor and physical development. 3.3. Molecular analysis

Twelve mutations were identified in the 8 patients (Table 2); out of 4. Discussion the twelve mutations, only c.659C > TintheBCKDHA gene has been previously reported (Rodriguez-Pombo et al., 2006). 11 of the muta- In this study, we presented 7 Chinese MSUD patients, and tions were novel. 5 of the novel mutationsdc.178G > T, c.491T > C, described their symptoms, clinical and imaging findings, treatment c.740A > G, c.1214_1219dupCCAACC and IVS6 þ 1delGdwere found and response. This condition has been rarely reported in this in the BCKDHA gene in patients 1e4. 5 of the novel population, so the findings will be highly useful for future cases of

Table 2 Mutations detected in eight Chinese patients with MSUD.

Patients Gene Mutation at nucleotide Mutation type Mutation at PolyPhen-2.0 Mutationtaster Conservation Frequency References level protein level prediction and prediction and score score

1 BCKDHA IVS6 þ 1delG Heterozygous splicing N/A N/A N/A 1/16 This study BCKDHA c.740A > G Heterozygous H247R Probably Disease causing Yes 1/16 This study damaging (0.99) (0.99) 2 BCKDHA c.659C > T Heterozygous A220V Probably Disease causing Yes 1/16 (Rodriguez-Pombo et al., damaging (1) (0.99) 2006; Park et al., 2011) 3 BCKDHA c.178G > T Heterozygous E60X N/A Disease causing (1) Conserved in 2/16 This study mammals BCKDHA c.1214_1219dupCCAACC Heterozygous P405_N406 N/A Disease causing (1) No 1/16 This study dup 4 BCKDHA c.178G > T Heterozygous E60X N/A Disease causing (1) Conserved in 2/16 This study mammals BCKDHA c.491T > C Heterozygous L164P Probably Disease causing Yes 1/16 This study damaging (0.99) (0.99) 5 BCKDHB IVS4,2A > C Homozygous splicing N/A N/A N/A 2/16 This study 6 BCKDHB c.767A > G Heterozygous Y256C Probably Disease causing Yes 1/16 This study damaging (1) (0.99) BCKDHB c.768C > G Heterozygous Y256X N/A Disease causing (1) Yes 1/16 This study 7 BCKDHB c.482T > G Heterozygous V161G Probably Disease causing Partly 1/16 This study damaging (0.99) (0.99) conserved BCKDHB c.508C > T Heterozygous R170C Probably Disease causing Partly 1/16 This study damaging (1) (0.99) conserved 8 DBT c.1A > G Homozygous M1V Benign (0) Polymorphism Yes 1/16 This study (0.98) X. Li et al. / European Journal of Medical Genetics 58 (2015) 617e623 621

Table 3 Amino acid alignment in the BCKDC E1a subunit residues (highlighted in bold).

Table 4 Amino acid alignment in the BCKDC E1b and E2 subunit residues (highlighted in bold).

MSUD in the Chinese population. Further, we found 11 novel mu- been reported (http://www.hgmd.cf.ac.uk/ac/index.php). The tations associated with MSUD. founder mutation, c.1312T > AinBCKDHA, shows a carrier fre- The majority of MSUD patients have the classical type (Strauss quency of up to 1 in 10 alleles in certain Mennonite populations in et al., 1993). This was true in our study too, as 6 of the 8 patients theUS(Puffenberger, 2003). Patients harboring this mutation are presented with typical phenotypes of classic MSUD. Moreover, only thought to have Mennonite ancestry (Love-Gregory et al., 2002). 1 patient (Patient 6) had intermediate MSUD. Patient 7 was diag- Some Gypsy-origin patients with classical MSUD have been re- nosed by newborn screening. She has not exhibited any symptoms ported to harbor the BCKDHA mutation c.117delC. This mutation is to date and therefore her MSUD type has not been classified. regarded as a founder mutation, with an estimated incidence rate Brain edema is a typical finding in the cranial MRI and CT scans of 1.4% among healthy transients from South Portugal (Quental of MSUD patients. The cranial MRI findings are always normal in et al., 2009). c.538G > CinBCKDHB, with a carrier frequency of 1 the first few days of life. After the edema disappears, damage in in 113 alleles is regarded as the founder mutation in the Ashkenazi brain regions such as globus pallidus and the brainstem has been Jewish population (Edelmann et al., 2001). No founder mutation reported (Brismar et al., 1990). In our study, 7 in 8 patients showed has been detected in the Chinese population. In this study, twelve damaged basal ganglia area especially in globus pallidus which mutations were found in 8 Chinese patients. Out of the twelve were typical in MSUD patients. Only one patient's cerebral MRI BCKDHA mutations, only c.659C > T has been reported before. revealed leukodystrophy and corpus callosum dysplasia. Also, Rodríguez-Pombo detected c.659C > Tinapatientwithclassical damaged frontal lobes, brainstem, insular cortex and orpora MSUD (Rodriguez-Pombo et al., 2006). It has also been found in quadrigemina were observed. Brain edema was not observed which Korean patients (Park et al., 2011). 11 novel mutations were probably because the cerebral MRI was taken in relatively late identified in this study. 2 intron mutations, IVS6 þ 1delG in course of the disease. BCKDHA and IVS4,2A > CinBCKDHB, may affect protein splicing. MSUD is initially diagnosed based on the clinical features and 1 duplication mutation, c.1214_1219dupCCAACC in BCKDHA,and2 the peculiar maple syrup odor of the urine. Blood BCAAs analysis is premature termination codon mutations, c.178G > TinBCKDHA the most convenient method, especially in newborn screening. If and c.768C > GinBCKDHB, may also result in a major change in the this patient has high blood alloisoleucine (>5 mm/L), MSUD should protein sequence. The other 5 were novel missense mutations, be considered (Schadewaldt et al.,1999). Urine organic acid analysis which were conserved or partly conserved in several species. is helpful for the diagnosis and differential diagnosis of other Although the c.1A > GmutationintheDBT gene was predicted to organic acidurias. Further, enzyme assay and genetic studies are be benign, no further mutations were detected in the other 4 important for confirming the diagnosis. In this study, except one genes; thus, a pathogenic basis cannot be ruled out. Only 1 het- patient who died before diagnosis, the remaining 7 patients had erozygous mutation, c.659C > TinBCKDHA, was detected in Pa- typical findings from the blood amino acids and urine organic acids tient 2, the other mutation might be a large deletion or analyses. duplication. To date, more than 200 mutations associated with MSUD have MSUD patients require life-long dietary restriction and strict 622 X. Li et al. / European Journal of Medical Genetics 58 (2015) 617e623 monitoring of the BCAAs levels in order to avoid brain damage laboratory of molecular diagnosis and study on pediatric genetic (Morton et al., 2012). Early diagnosis and treatment are crucial to diseases and the 12th Five-year Plan National Key Technology R & D reduce the severity of brain injury (Strauss et al.,1993). In our study, Program from the Ministry of Science and Technology (No. only 1 patient who was detected by newborn screening had normal 2012BAI09B04). development. Psychomotor retardation was observed in the remaining 6 patients, although they responded well to the treat- Conflicts of interest ment. With the improvement of newborn screening using LC- MSMS in mainland China, an increasing number of MSUD pa- This work has fully complied with research ethics, and abide by tients would get early dietary or medical intervention and normal the Code of Ethics of the World Medical Association. All the authors development. declared that they have no conflicts of interest to this work. The treatments for MSUD include low protein diet; supple- mentation with BCAAs-free formula; and symptomatic treatment Acknowledgments during metabolic crises, such as mannitol intake to cure encepha- ledema, insulin injection to lower the blood glucose level, and use Thanks for the cooperation of all patients and physicians of of D-carbamylglutamate to reduce the blood ammonia level (Kalkan Peking University First Hospital. We also thank Synutra Interna- et al., 2009; Yoshino et al., 1999). As the liver is responsible for 15% tional Inc. for their support. of BCKDC production, transplantation with a liver from a donor with normal BCKDC activity can restore some enzymatic activity in References MSUD patients (Diaz et al., 2014; Strauss et al., 2006). Liver transplantation could benefit classical MSUD patients by arresting brain Brismar, J., Aqeel, A., Brismar, G., Coates, R., Gascon, G., Ozand, P., 1990. Maple syrup damage, although it does not reverse the disease process (Diaz urine disease: findings on CT and MR scans of the brain in 10 infants. AJNR Am. J. Neuroradiol. 11 (6), 1219e1228. et al., 2014). MSUD patients with a structurally normal liver could Chuang, D.T., Chuang, J.L., Wynn, R.M., 2006. Lessons from genetic disorders of also donate their liver to patients who do not have BCKDC defects. branched-chain amino acid metabolism. J. Nutr. 136 (1 Suppl. l), 243Se249S. Domino liver transplantation is also possible, and it was first per- Diaz, V.M., Camarena, C., de la Vega, A., Martinez-Pardo, M., Diaz, C., Lopez, M., et al., 2014. Liver transplantation for classical maple syrup urine disease: long-term formed in 2014 (Feier et al., 2014). L-carnitine has been reported to follow-up. J. Pediatr. Gastroenterol. Nutr. 59 (5), 636e639. benefit MSUD patients by preventing oxidative damage to the cells Dodd, P.R., Williams, S.H., Gundlach, A.L., Harper, P.A., Healy, P.J., Dennis, J.A., et al., (Guerreiro et al., 2015). L-carnitine can also prevent in vitro DNA 1992. Glutamate and gamma-aminobutyric acid neurotransmitter systems in the acute phase of maple syrup urine disease and citrullinemia encephalopa- damage in human peripheral leukocytes (Mescka et al., 2014). e e thies in newborn calves. J. Neurochem. 59 (2), 582 590. Further, norleucine, which competes with leucine in the blood - Edelmann, L., Wasserstein, M.P., Kornreich, R., Sansaricq, C., Snyderman, S.E., brain barrier (Tews et al., 1991), was considerably effective in Diaz, G.A., 2001. Maple syrup urine disease: identification and carrier-frequency deferring encephalopathy and increasing the survival rate in mouse determination of a novel founder mutation in the Ashkenazi Jewish population. Am. J. Hum. Genet. 69 (4), 863e868. models of intermediate MSUD and classic MSUD (Zinnanti et al., Feier, F.H., Miura, I.K., Fonseca, E.A., Porta, G., Pugliese, R., Porta, A., et al., 2014. 2009). In our study, except Patient 5 who died before diagnosis, Successful domino liver transplantation in maple syrup urine disease using a e the other 7 patients were treated by L-carnitine; vitamins B1, B2, related living donor. Braz. J. Med. Biol. Res. 47 (6), 522 526. Fu, X., Iga, M., Kimura, M., Yamaguchi, S., 2000. Simplified screening for organic and B12, L-arginine; and glucose infusion when needed. Low pro- acidemia using GC/MS and dried urine filter paper: a study on neonatal mass tein diet with a leucine-, valine- and isoleucine-free formula was screening. Early Hum. Dev. 58 (1), 41e55. given to patients. 5 patients showed clinical improvement, Guerreiro, G., Mescka, C.P., Sitta, A., Donida, B., Marchetti, D., Hammerschmidt, T., et al., 2015. Urinary biomarkers of oxidative damage in Maple syrup urine including fewer seizures attacks, progresses in response and disease: the l-carnitine role. Int. J. Dev. Neurosci. 42C, 10e14. movement. 1 patient (Patient 7) was detected by newborn Hamerton, J.L., 1982. Clonal culture of human amniocytes. Am. J. Med. Genet. 13 (3), screening who was very healthy which indicated the disease was 351. under control. 1 patient (Patient 5) did not presented with obvi- Harper, P.A., Healy, P.J., Dennis, J.A., 1990. Maple syrup urine disease (branched chain ketoaciduria). Am. J. Pathol. 136 (6), 1445e1447. ously progress after treatment. Huang, Y., Zielke, H.R., Tildon, J.T., Zielke, C.L., Baab, P.J., 1996. Elevation of amino Prenatal diagnosis is an important approach to identify birth acids in the interstitial space of the rat brain following infusion of large neutral defects, especially in conditions that are difficult to treat. Definite amino and keto acids by microdialysis: leucine infusion. Dev. Neurosci. 18 (5e6), 415e419. genetic analysis of the proband has enabled DNA-based prenatal Jouvet, P., Kozma, M., Mehmet, H., 2000. Primary human fibroblasts from a maple diagnosis of single-gene disorders. You et al. reported a Chinese syrup urine disease patient undergo apoptosis following exposure to physio- family affected by MSUD associated with BCKDHA mutations; pre- logical concentrations of branched chain amino acids. Ann. N. Y. Acad. Sci. 926, 116e121. natal diagnosis for the fetus was performed (You et al., 2014). In this Kalkan, U.S., Coker, M., Habif, S., Saz, E.U., Karapinar, B., Ucar, H., et al., 2009. The first study, too, prenatal diagnosis for a fetus from a family with BCKDHB use of N-carbamylglutamate in a patient with decompensated maple syrup gene mutations was successfully carried out. Only 1 heterozygous urine disease. Metab. Brain Dis. 24 (3), 409e414. > fi Killian, D.M., Chikhale, P.J., 2001. Predominant functional activity of the large, mutation, IVS4, 2A CinBCKDHB, was identi ed. Elevated a-keto neutral amino acid transporter (LAT1) isoform at the cerebrovasculature. acids were not detected in the amniotic fluid of the mother. Post- Neurosci. Lett. 306 (1e2), 1e4. natal analysis of blood amino acids and normal development of the Kimura, M., Yamamoto, T., Yamaguchi, S., 1999. A personal computer-based system fi for interpretation of gas chromatography mass spectrometry data in the diag- infant con rmed the prenatal diagnosis. nosis of organic acidaemias. Ann. Clin. Biochem. 36 (Pt 5), 671e672. In conclusion, this study reports 11 new mutations associated Love-Gregory, L.D., Grasela, J., Hillman, R.E., Phillips, C.L., 2002. Evidence of common with MSUD in the Chinese population, which will help with the ancestry for the maple syrup urine disease (MSUD) Y438N allele in non- e genetic diagnosis of this disease in the future. To investigate the Mennonite MSUD patients. Mol. Genet. Metab. 75 (1), 79 90. Marshall, L., DiGeorge, A., 1981. Maple syrup urine disease in the old order Men- mutation spectrums of disease-causing genes associated with nonites. Am. J. Hum. Genet. 33, 139A. MSUD of Chinese population, a large-scale study in China should be Menkes, J.H., Hurst, P.L., Craig, J.M., 1954. A new syndrome: progressive familial necessary. infantile cerebral dysfunction associated with an unusual urinary substance. Pediatrics 14 (5), 462e467. Mescka, C.P., Wayhs, C.A., Guerreiro, G., Manfredini, V., Dutra-Filho, C.S., Vargas, C.R., Funding source 2014. Prevention of DNA damage by L-carnitine induced by metabolites accu- mulated in maple syrup urine disease in human peripheral leukocytes in vitro. Gene 548 (2), 294e298. This work was supported by the grants from the National Nat- Morton, D.H., Strauss, K.A., Robinson, D.L., Puffenberger, E.G., Kelley, R.I., 2012. ural Science Foundation of China (No. 81471097), Beijing key Diagnosis and treatment of maple syrup disease: a study of 36 patients. X. Li et al. / European Journal of Medical Genetics 58 (2015) 617e623 623

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