Evolution of maple syrup urine disease in patients diagnosed by newborn screening versus late diagnosis

e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 5 ) 1 e8 Official Journal of the European Paediatric Neurology Society Original article Evolution of maple syrup urine disease in patients diagnosed by newborn screening versus late diagnosis M.L. Couce a,*, F. Ramos b, M.A. Bueno c, J. Dı́az d, S. Meavilla e,
ndez-Marmiesse a, A. Garcı́a-Cazorla b
veda a, A. Ferna M.D. Bo a Metabolic Unit, Servei of Neonatology, Department of Pediatrics, Hospital Clı́nico Universitario de Santiago, IDIS, CIBERER, ISCIII, Santiago de Compostela, Spain b Department of Neurology, Neurometabolic Unit, Hospital Sant Joan de D
eu, and CIBERER, ISCIII, Barcelona, Spain c Metabolic and Dismorphology Unit, Department of Pediatrics, Hospital Universitario Virgen del Rocı́o, Sevilla, Spain d Gastroenterolgy, Nutrition and Metabolic Unit, Hospital Central de Asturias, Spain e Department of Gastroenterology and Nutrition, Metabolic Unit, Hospital Sant Joan de D
eu, Barcelona, Spain article info abstract Article history: Maple syrup urine disease (MSUD) is a rare metabolic disorder for which the newborn Received 28 February 2015 screening (NBS) is possible but it has not been yet implemented for most Spanish regions. Received in revised form In the present study, we assess the clinical features and outcome of 14 MSUD Spanish 14 June 2015 patients with similar treatment protocol diagnosed either by NBS or by clinical symptoms. Accepted 2 July 2015 Eight patients were detected by NBS, four classic and four moderate MSUD. The average age at detection was 4.6 days, the mean plasmatic concentration of leucine at diagnosis Keywords: was 1807 mM; the average number of days with leucine >1000 mM was 0.7 (0e4) and the MSUD mean number of total hospitalizations was 1.6 (0e5). Mean follow-up time was 70 months. Outcome They had good evolution: all remain asymptomatic, but 2 patients have attention deficit Leucine and hyperactivity disorder. Six patients with late diagnosis of classic MSUD were followed Newborn screening during 41 months. All presented with acute encephalopathy during the first month of life, Mood disorders mean leucine levels of 2355 mM, mean number of days with leucine >1000 mM of 6.6 (1e13) Encephalopathy and mean number of total hospitalizations of 5.3 (4e7). Only two patients have a psychomotor development index in the lower limit (80 and 83). For all patients a good genotype ephenotype correlation was found and four novel mutations were identified: p.A311H, p.T84S, p.T397L, pL398P. Our study support that NBS improves prognosis of MSUD patients. But early diagnosis and an aggressive treatment together with a close monitoring of leucine levels improve neurological evolution in MSUD patients, even for those not detected by NBS. © 2015 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved. * Corresponding author. Metabolic Unit, Servei of Neonatology, Department of Pediatrics, Hospital Clı́nico Universitario de Santiago, IDIS, CIBERER, ISCIII, A Choupana s/n, 15706 Santiago de Compostela, Spain. Tel.: þ34 981950162. E-mail address: maria.luz.couce.pico@sergas.es (M.L. Couce). http://dx.doi.org/10.1016/j.ejpn.2015.07.009 1090-3798/© 2015 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved. Please cite this article in press as: Couce ML, et al., Evolution of maple syrup urine disease in patients diagnosed by newborn screening versus late diagnosis, European Journal of Paediatric Neurology (2015), http://dx.doi.org/10.1016/j.ejpn.2015.07.009 2 1. e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 5 ) 1 e8 Introduction Maple syrup urine disease (MSUD, OMIM 248600) is a rare metabolic disorder of autosomal recessive inheritance caused by deficiency in the activity of the branched-chain a-ketoacid dehydrogenase complex (BCKD, E.C.1.2.4.4.), which catalyzes the oxidative decarboxylation of the branched-chain keto acids in the second step from catabolic pathway of the branched chain amino acids (BCAAs) (leucine, isoleucine and valine). BCKD is a multi-enzyme complex comprised of three catalytic components: E1, a decarboxylase composed of E1a and E1b subunits which requires thiamine pyrophosphate as a coenzyme; E2, a homo-24-meric-transacylase; and E3, a dihydrolipoamide dehydrogenase.1 A deficiency of E1 or E2 component can cause MSUD whereas a deficiency of the E3 component produces a specific syndrome (dihydrolipoamide dehydrogenase deficiency) with congenital lactic acidosis.2 Traditionally, the metabolic phenotype of MSUD on the basis of residual BCKD enzyme activity is termed classic (<3%) or intermediate (3e30%). Rarely, affected individuals have partial BCKD that only manifests intermittently or responds to dietary thiamine therapy. In the classical MSUD (75% of cases), clinical onset usually occurring within the first weeks after birth, including a maple syrup odor, acute metabolic decompensation with feeding problems and drowsiness, followed by progressive coma with involuntary movements, seizures and respiratory failure. The diagnosis is usually established by measuring plasma BCAA levels including alloisoleucine, which is pathognomonic for the disorder, and their corresponding BCKAs in urine. Treatment consists of dietary leucine restriction, BCAAfree medical foods, judicious supplementation with isoleucine and valine, and frequent clinical and biochemical monitoring, attending for a possible metabolic decompensation.3,4 Phenylbutyrate therapy for maple syrup urine disease may be a valuable treatment during the acute phase.5 The main goal is to maintain leucine levels below 200 mmol/L within the first 6 years,38 and there after keep up those levels below 300 mmol/L. Orthotropic liver transplantation can be an effective therapy for classic MSUD.6,7 Leucine and 2-ketoisocaproic acid appear to be the most neurotoxic metabolites.8e10 The occurrence of oxidative stress in MSUD, probably secondary to the high production of free radicals and low total oxidant status during treatment, also contributes to the neurological sequelae present in most patients.11e13 An estimated prevalence of 1 in 185,000 newborns has been found.1 However, in certain communities there is an overexpression of this entity, such as the Mennonite14,15 and Galician (North-West of Spain) populations where the reported incidence was 1 in 52,541 newborns.16 To date, over 160 disease-causing mutations have been detected among the three different genes encoding for the BCKD components, BCKDHA (E1a subunit), BCKDHB (E1b subunit) and DBT (E2 subunit) (Human Gene Mutation Database, http://www.hgmd. cf.ac.uk).17e19 Expanded newborn screening by tandem mass spectrometry detects MSUD by measuring the whole blood combined leucineeisoleucine concentration and its ratio to other amino acids (AA) such as alanine and phenylalanine. The clinical evolution of patients detected by NBS seems to be favorable in most cases,20e22 but not always23 and long-term evolution is still doubtful. We present the genotype, phenotype and the follow up data of the children with MSUD diagnosed in four regions of Spain in the last twelve years, either by NBS or by clinical symptoms. 2. Methods 2.1. Study population The present study population comprised MSUD patients diagnosed in four of the Spanish regional (Galicia, Asturias, ~ a and western Andalusia) NBS programs or by clinical Catalun symptoms. The period of study was from January of 2001 to December of 2013. At diagnosis, the following parameters were evaluated: age, familiar consanguinity, deceased siblings with similar symptoms, results of dried blood spots obtained in newborn screening and/or plasma concentrations of BCAAs and alloisoleucineto detection, presence or absence of clinical symptoms, plasma AA concentrations in the beginning of the treatment with maximum peak for leucine, the need of dialysis measures, the days with leucine concentration above 1000 mM. Diagnosis was confirmed by BCKD activity and/or mutation analysis of the BCKDHA/BCKDHB/DBT genes. Treatment was held according to the Spanish MSUD Guidelines,24 during follow-up patients received not only a dietary leucine restriction, according to age and tolerance, but also valine and isoleucine supplementation. Thiamine (100e300 mg per day) and supplement of vitamins A and E was also administered. Micronutrient profile was analyzed annually providing specific mineral and/or vitamin supplement if deficiencies were detected. The main goal was to maintain leucine concentrations below 300 mmol/L and, in children under 6 years old, keep this level in 200 mmol/L, with isoleucine and valine levels between 200 and 400 mmol/L, controlling the normal range concentrations of glutamine, alanine, tryptophan, tyrosine, methionine and the ratios Leu/ Tyr and Leu/Ala. During an acute intercurrent illness the treatment protocol was carefully managed with cessation or reduction of protein intake to 50% for 24e48 h, depending on the severity of the illness, whilst providing a high energy intake with an extra 20% of caloric requirements through carbohydrates, lipids and double dose of carnitine, valine and isoleucine. In case of vomiting or clinical deterioration, an urgent hospital admission for intravenous glucose infusion without branched-chain amino acids was recommended. Clinical course was subsequently monitored. Follow-up included measurement of AAs in blood spot/plasma, with individual yearly median values of leucine and maximum leucine concentrations, IQ testing and gross motor function measurements. Possible associations between leucine levels, maximum concentration of leucine and type of diagnosis, Please cite this article in press as: Couce ML, et al., Evolution of maple syrup urine disease in patients diagnosed by newborn screening versus late diagnosis, European Journal of Paediatric Neurology (2015), http://dx.doi.org/10.1016/j.ejpn.2015.07.009 e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 5 ) 1 e8 clinical findings, neuroimaging and IQ outcome were assessed. Informed consent was obtained from the parents of all patients. The study was approved by the Ethics Committee of each Hospital. 2.2. Analytical methods - NBS: Amino acids analysis from dried blood spot sampling was performed by MS/MS. MSUD was suspected in any newborn with an increase in combined leucineeisoleucine concentration higher than the 99.9th percentile of neonatal population (cut off <380 mM). - Quantitative analysis of amino acids in plasma samples was carried out by ion-exchange chromatography (Biochrom 30 autoanalyser) after deproteinization of the sample (plasma or urine) with 5-sulfosalicylic acid, with post-column reaction with ninhydrin, and making use of Lnorleucine as an internal standard. - The analysis of branched-chain amino acids e leucine, isoleucine and valine e is also made from dried blood spot sampling by ion exchange chromatography with chromatographic conditions and instrument similar to the plasma sample, but with a previous step of elution and deproteinization with trichloroacetic acid 3%. - BCKD activity: It was measured the decarboxylation of the marked leucine ([1-14C] leucine) and cultured fibroblasts with an intraassay control. - Molecular testing: DNA was isolated and sequenced by standard procedures for blood samples of all patients and their parents, except for one patient conceived by in vitro fertilization with oocyte donation. Molecular analysis of BCKDHA/BCKDHB/DBT was performed using standard procedures. Primers were designed to overlap the coding sequences and their flanking regions (sequences available on request). PCR products were purified by ExoSap (usb®) enzyme and sequenced using a Big Dye Terminator Cycler Sequencing Ready reaction kit and the manufacturer's protocol (Applied Biosystems). The sequencing reactions were performed in an ABI 3130XL Genetic Analyser. - Statistical analysis: To determine the possible correlation of variables studied we used the Wilcoxon signed-rank test if one of the variables was quantitative and the other qualitative since the sample size was insufficient to presume the normality in the data. The Fisher's exact test was performed if both were quantitative (again, the limited sample size didn't allow us to apply the Pearson's Chisquared test), and the Pearson product-moment correlation coefficient if both were qualitative. 2.3. Follow-up Cognitive function was assessed by psychomotor development index (PDI) or intellectual quotient (IQ) using the Wechsler Intelligence Scale (WISC R) for school-age children, the McCarthy Scales of Children's Abilities (MSCA) for zine scale in inpreschool-age children, and the Brunet-Le fants. The overall index score of PDI or IQ is considered in the normal range when it is above 85. 3 The gross motor function measure (GMFM) was performed for all children in the last year of evolution.25 Levels of writing, reading and mathematical calculation were assessed establishing these values: 0 (no trouble); 1 (mild trouble); 2 (moderate trouble) and 3 (profound trouble). MRI Siemens Magnetom Symphony Master Class 1.5 T was employed for brain MRI. 3. Results 3.1. Diagnosis We evaluated 14 patients with MSUD, with the following geographical distribution: 6 cases from Galicia, 1 case from ~ a and 3 from Andalusia. Asturias, 4 from Catalun All patients were diagnosed in the first 20 days of life (Table 1). Eight of them (p1ep8) were early detected by NBS, being average analytical sample collection time 4.6 days (range: 2e8) and presented a Leu þ Ile mean level of 1253 mM (range: 514e3376; cutoff 380 mM). The average age for diagnosis was 9.7 days (range 7e19). The mean plasmatic concentrations at diagnosis of BCAA were: leucine, 1807 mM (range 263e2500), valine: 684 mM (range 161e1031), and isoleucine: 489 mM (range 80e1437). Three patients with moderate phenotype (p4, p5, p8) were asymptomatic but five (four classic and 1 moderate MSUD) presented clinical symptoms like poor feeding, irritability, lethargy, coma in p2 and p6 and also sweet odor smelling in three patients. Since the treatment started (dietetic, thiamine, and dialysis in the four patients with marked symptoms of intoxication), the leucine levels remained above 1000 mM on average for 2 days (0e4 days). Brain MRI was performed for the two comatose patients (p2, p6) showing large areas with cytotoxic edema which affects corticospinal tracts, thalamus, brainstem and the white matter of the cerebellum. In other six patients (p1,3,4,5,7,8) ultrasonography showed no signs of cerebral edema. In the cases p9ep14 (LD) the diagnosis was suspected by clinical symptoms such as lethargy, irritability, drowsiness, and stereotyped movements such as “fencing”, “bicycling” and reached coma in 50% of cases. Two cases showed sweet odor smelling. The average age of detection was 11.5 days (range 9e14), showing at diagnosis a mean value of leucine levels of 2355 mM (range 1600e3241). The phenotype was considered classic in all of them. After the initiation of the treatment (in all of them hemodiafiltration was performed) leucine levels remained above 1000 mM during 2.96 days (range 1e5 days). Brain MRI was performed in 5 of the 6 patients resulting altered in all of them, showing edema in the cerebellum, brainstem, cerebral peduncle, white matter and pale corticospinal tract with marked restriction of diffusion. All the mutations found in the 14 patients are summarized in Table 2, being 8 patients homozygous for one mutation and 6 patient's compound heterozygous. Consanguinity was shown in 5 cases (35.7%) and 15 different mutations were detected. Seven patients had mutations in BCKDHB gene, 3 in BCKDHA gene and 3 in the DBT gene. All mutations were previously described26e31 except four. The four novel mutations identified (p.D311H in BCKDHB gene and p.T84S, p.T397L, pL398P in DBT gene) were evaluated with in silico prediction Please cite this article in press as: Couce ML, et al., Evolution of maple syrup urine disease in patients diagnosed by newborn screening versus late diagnosis, European Journal of Paediatric Neurology (2015), http://dx.doi.org/10.1016/j.ejpn.2015.07.009 4 e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 5 ) 1 e8 Table 1 e Characteristics of patients with MSUD at the time of detection and the evolutional IQ, leucune levels and hospital admissions. Patient Sex 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Way of Leu þ Ilea (mM) Time of sample Time of diagnosis Leub (mM) Ileb (mM) Valb (mM) Poor feeding detection collection (days) (days) M M F F M M M M M F F F F F NBS NBS NBS NBS NBS NBS NBS NBS LD LD LD LD LD LD 1467 3376 590 514 NA 1124 565 1141 7 7 2 3 4 3 8 3 14 7 7 8 19 7 9 7 14 12 9 9 13 10 1682 2500 2269 263 1605 2250 2186 1704 1671 2480 2425 2717 1600 3241 386 412 439 101 80 528 530 1437 754 511 205 267 208 458 907 893 926 280 161 1031 679 596 581 828 301 383 404 501 þ þ þ þ þ þ þ þ þ þ þ M: male; F: female; NBS: MSUD identified by newborn screening; LD: late diagnosis (MSUD patients identified on clinical grounds); PD: peritoneal dialysis, H: hemodiafiltration. a Combined leucineeisoleucine concentration measured in DBS by tandem mass spectrometry. b Quantitative analyses carried out by ion-exchange chromatography. software's to assess the possible functional impact of the mutation (Table 3). The enzymatic activity was measured in 5 patients detected by NBS. In all of them enzymatic levels correlate with the clinical phenotype and with the phenotype associated to the mutations found (Table 1), detecting an enzymatic activity above 3% in the three patients with moderate phenotype meanwhile the two patients with classic phenotype showed an enzymatic activity less 3%. Cases whose enzymatic analyses were not performed the clinical phenotype also correlated with the phenotype predicted by mutations found. 3.2. Follow-up The average follow-up period was 4 years and 10 months (range 11 monthse12 years). All cases were non-responsive to thiamine. Lifetime leucine levels are reflected on Fig. 1; in patients p4, p5 and p7 (two of them with moderate phenotype and one classic) leucine levels remained below or close to 200 mM in the first years of life meanwhile for the other classic cases leucine levels range from 124 to 345 mM, except for patients p9 and p10 with the poorest control. Table 1 reflects the decompensation episodes, showing for each patient the number of hospital admissions, number of days admitted in the hospital and the number of days with leucine levels above 1000 mM. We can observe that patients diagnosed by NBS have had indirect data of better quality of life (mean number of acute metabolic decompensations and mean number of total hospitalizations: 0.7 and 1.6, versus 6.6 and 5.3 respectively). Patients p9 and p10 had more days (13 and 10) with inadequate leucine levels, requiring dialytic measures (7 and 4 times respectively). Given the limited number of patients it is difficult to achieve statistically significant differences in most of the comparisons performed between the two groups (NSB and LD), but it was found a significant difference for the number of days of hospitalization (NSB: 8.75 vs LD: 53 days; p: 0.005) as well as for days with leucine >1000 mM (NSB: 0.85 vs LD: 6.63 days; p-value: 0.001). General clinical outcome was positive in all patients. Control MRI was performed in 2 patients with late diagnosis (p9, p10) being normal and in the others it was not considered for its good performance. Motor function assessed by GMFM was normal in all of them. The PDI/IQ index was above 80 in all the patients (Table 1). The mean PDI/IQ index was 97 (85e117) for NBS detected patients. In two patients (p9, p10) with late diagnosis the PDI/IQ index was 80 and 83 respectively and they have index 11 points below their healthy siblings (10e12). Five patients attending school were followed for more than 5 years. No learning difficulties were observed, but two of them suffer from attention deficit and hyperactivity disorder (ADHD) who were treated with extended-release methylphenidate presenting a slight improvement in its evolution. 4. Discussion This study analyzes clinical, biochemical and genetic data from a heterogeneous group of MSUD patients from 4 Spanish hospitals regarding phenotype (10 classic, 4 moderate) and disease detection (NBS and/or clinical symptoms), which allows conclusions to be drawn on potential factors which could influence MSUD patients outcome. Increased plasma concentration of leucine and its metabolite, a-ketoisocaproic acid, is associated with the appearance of neurological symptoms.1,32 Neonatal encephalopathy has been associated with a fourfold higher risk of global functional impairment.33 One MSUD patient detected by NBS was initially asymptomatic but developed quickly cerebral edema, Please cite this article in press as: Couce ML, et al., Evolution of maple syrup urine disease in patients diagnosed by newborn screening versus late diagnosis, European Journal of Paediatric Neurology (2015), http://dx.doi.org/10.1016/j.ejpn.2015.07.009 5 e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 5 ) 1 e8 Irritability/ Stereotyped Coma Sweet odor Cerebral Dialysis lethargy movements smelling edema þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ Psychomotor Hospital Days admitted Leu development admissions to hospital >1000 index/intellectual (number) mM (days) quotient (age at testing) 117 (9 y) 89 (8 y) 104 (6 y) 108 (4 y) 92 (2 y) 88 90 (1 y) 93 80 83 85 85 85 85 e PD PD e e H H e H H H H H H advising an early aggressive treatment in any patient with a positive NBS for MSUD.23 In our study, a high percentage (62.5%) of patients detected by NBS showed clinical symptoms of intoxication with elevated levels of leucine (>1500 mM) at the moment of detection, although encephalopathy with signs of cerebral edema and coma was detected in 2 out of 8 patients. One of these 2 patients (p2) was detected in 2001 when screening was advised in older age (5e8 days) showing the importance of early NBS. Only 2 out of 6 (p9,p10) patients with late diagnosis have a PDI/IQ below the normal range despite all of them showed encephalopathy at detection and even the 5 most affected had 5 5 1 0 0 2 0 0 7 5 6 5 4 5 19 38 11 0 0 2 0 0 90 62 46 34 32 54 1 0 1 0 0 4 0 0 13 10 6 1 2 8 cerebral edema. This is probably due to and early diagnosis and treatment, unlike other series.34 It is well known that transient period of MSUD encephalopathy appears fully reversible, triggering no global or focal ischemic brain damage. In addition, measures of PDI/IQ correlated inversely with average lifetime plasma leucine and its concentration ratio to valine and directly correlated to the frequency of amino acid monitoring since prolonged amino acid imbalances, particularly if they occur during the early years of brain development, lead to structural and functional neurologic abnormalities that have morbid long-term psychomotor consequences.33,35 In fact, in these two patients (p9, p10) with higher levels of Table 2 e Biochemical and genetic characteristics of MSUD patients (reference sequence for BCKDHA: NM_000709; BCKDHB: NM_000056; DBT gene: NM_001918). Patient 1 Enzymatic activity 4 Clinical phenotype Gene Nucleotide change Amino acid change M BCKDHA (Ia) c.853G>C c.659C>T c.348delA c.514G>T c.641T>A c.641T>A c.931G>C c.348delA c.595_596 delAG c.251G>C c.1385G>C c.659C>T c.117delC c.117delC c.1190C>T c.1192C>T c.595_596delAG c.1150A>G c.503G>A c.970C>T p.Ala285Pro p.Ala220Val p.Lys116fs p.Gly172Trp p.Ile214Lys p.Ile214Lys p.Asp311His p.Lys116fs p.Pro200fs p.Trp84Ser p.Arg462Pro p.Ala220Val p.Arg40fs p.Arg40fs p.Thr397Ile p.Leu398Phe p.Pro200fs p.Ser384Gly p.Arg168His p.Arg324Stop 2 1.3 C BCKDHB (Ib) 3 4 1.2 26 C M BCKDHB (Ib) BCKDHB (Ib) 5 6 7 14 e e M C C BCKDHB (Ib) BCKDHB (Ib) DBT (E2) 8 9 10 11 e e e e M C C C BCKDHA (Ia) BCKDHA (Ia) BCKDHA (Ia) DBT (E2) 12 13 14 e e e C C C BCKDHB (Ib) DBT (E2) BCKDHB (Ib) References Wynn RM et al.27 Rodrı́guez-Pombo Rodrı́guez-Pombo Rodrı́guez-Pombo Rodrı́guez-Pombo Rodrı́guez-Pombo Novel Rodrı́guez-Pombo Henneke et al.28 Novel Nellis et al.29 Rodrı́guez-Pombo Rodrı́guez-Pombo Rodrı́guez-Pombo Novel Novel Henneke et al.28 Tsuruta et al.30 Rodrı́guez-Pombo McConnel et al.31 Phenotype predicted by mutations M et et et et et al.26 al.26 al.26 al.26 al.26 et al.26 et al.26 et al.26 et al.26 et al.26 C C M U M C U C M C C U C C C M: moderate; C: classic; U: phenotype predicted by mutations is unknown (mutation novel). Please cite this article in press as: Couce ML, et al., Evolution of maple syrup urine disease in patients diagnosed by newborn screening versus late diagnosis, European Journal of Paediatric Neurology (2015), http://dx.doi.org/10.1016/j.ejpn.2015.07.009 6 e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 5 ) 1 e8 Table 3 e In silico prediction of functional impact for novel mutations detected using CONDEL (CONsensus DELeteriousness) software. Gene BCKDHB DBT DBT DBT AA_CHANGE SIFT PPH2 MA D311H W84S T397I L398F 0 0 0 0.02 1 0.999 0.999 0.633 4.915 4.57 4.62 3.57 leucine in the follow-up, the PDI/IQ is in the lower limit of normal range (80 and 83 respectively) and they show a difference of at least 10 points with their healthy siblings. In the other patients leucine levels were generally maintained below 250 mM. The possibility of sending DBS directly from each patient's home to determine BCAAs levels and therefore to be frequently monitored was crucial. Patients should ideally monitor BCAAs levels once per week, which is usually not practical without a “send-in” filter paper method.3 In our study ADHD is present in 2 (n2, n3) out of 5 patients with more than 5 years of evolution and attending school. In other series, cumulative lifetime incidence of ADHD was 54% among individuals with MSUD on dietary therapy and MSUD patients with ADHD had a stronger positive correlation between plasma leucine levels and symptom severity compared with those without an ADHD diagnosis.33,36 In these patients, long-term neuropsychiatric assessment must be monitored.33,37 Neonatal encephalopathy is a strong predictor of neurological handicaps later in life. Detection of intellectual disability, ADHD or other behavioral and neuropsychiatric conditions needs longer term monitoring to achieve a proper diagnosis. It is known that patients who were encephalopathy at the time of diagnosis were five and ten times more likely, respectively, to later suffer from anxiety and depression.33 Interestingly, patient p1 with an enzyme activity of 4% (moderate phenotype) and without neonatal encephalopathy developed mood disorder (based on DSM-IV criteria) which responded favorably to standard antidepressants. It could be explained because he showed higher mean FATHMM 4.17 1.69 0.43 0.73 Score CONDEL_LABEL 0.79 0.69 0.78 0.66 Deleterious Deleterious Deleterious Deleterious leucine levels and BCAAs levels measurement performed much less frequently than recommended. In addition, it is well-established that oxidative stress contributes to brain damage in MSUD, and the use of appropriate antioxidants offers new perspectives for the prevention of the neurological damage in MSUD,11 but further research is necessary. Our patients, according to the control and treatment protocol they received vitamin complex with vitamin A, vitamin E and frequent intake of selenium. Surprisingly, four patients carrying mutations in the BCKDHA and BCKDHB genes presented the moderate phenotype, unlike data showed by others studies where classic phenotype and a worse clinical outcome were associated with BCKDHA gene.18 DBT mutation p.T84S occurs in the late nucleotide of exon 3 and the functional prediction provided by Human Splicing Finder software (http://www.umd.be/HSF/) is that such mutation disrupts the wild type donor site of exon 3, which will probably affect the correct splicing of mRNA. Indeed, patient 7 who carry this mutation have a classic clinical phenotype probably because the predicted splicing variant p.T84S is associated to p.Arg462Pro, also associated to classic phenotype in previous reports. The other three novel missense mutations (p.D311H, p.T397L and p.L398F) were evaluated with CONDEL software (http://bg.upf.edu/fannsdb/) that combines several bioinformatics tools: SIFT (Sorting Intolerant From Tolerant: http://sift.bii.a-star.edu.sg/); PPH2 (Polymorphism Phenotypingv2: http://genetics.bwh.harvard. edu/pph2/); MA (Mutation Assessor: http://mutationassessor. org/); FATHMM (Functional Analysis Through Hidden Fig. 1 e Mean and maximum leucine levels per year. Please cite this article in press as: Couce ML, et al., Evolution of maple syrup urine disease in patients diagnosed by newborn screening versus late diagnosis, European Journal of Paediatric Neurology (2015), http://dx.doi.org/10.1016/j.ejpn.2015.07.009 e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 5 ) 1 e8 Markov Models (v2.3): http://fathmm.biocompute.org.uk/) being all of them predicted as deleterious with important scores (Table 3). Patient 4, which carries the novel mutation p.D311H in BCKDHB gene was associated with moderate phenotype probably because the mutation placed in the other allele have an important residual activity (classified as moderateassociated in previous reports). The patient 11 which carries the two novel mutations p.T397L and p.L398F present a classical phenotype which indicates that probably both variants produce a DBT enzyme without any residual activity. In view of these results, we reinforce that prompt detection of leucine levels identified in the expanded newborn screening program promote a decline in the number of days with toxic leucine levels, and therefore are essential to improve neurological outcomes in MSUD patients. We also state that, in the cases detected by clinical symptoms, early detection and treatment since the onset of clinical symptoms, a close monitoring of BCAA levels in follow-up, and early and aggressive treatment in the face of any decompensation improves neurological outcome in these patients. Conflict of interest None of the authors have any conflict of interest to declare. references 1. Chuang DT, Shih VE. Maple syrup urine disease (branchedchain ketoaciduria). In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The metabolic and molecular basis of inherited disease. 8th ed. New York: McGraw-Hill; 2001. p. 1971e2006. 2. Grafakou O, Oexle K, van den Heuvel L, Smeets R, Trijbels F, Goebel HH, Bosshard N, Superti-Furga A, Steinmann B, Smeitink J. Leigh syndrome due to compound heterozygosity of dihydrolipoamide dehydrogenase gene mutations. Description of the first E3 splice site mutation. Eur J Pediatr 2003;162:714e8. 3. Strauss KA, Puffenberger ED, Morton D. Maple syrup urine disease. In: Pagon R, Adam M, Bird T, et al., editors. GeneReviews. Seattle, WA: University of Washington; Jan. 30, 2006. p. 1993e2013 [updated 2013 May 9]. 4. Frazier DM, Allgeier C, Homer C, Marriage BJ, Ogata B, Rohr F, Splett PL, Stembridge A, Singh RH. Nutrition management guideline for maple syrup urine disease: an evidence- and consensus-based approach. Mol Genet Metab 2014;112:210e7. 5. Brunetti-Pierri N, Lanpher B, Erez A, Ananieva EA, Islam M, Marini JC, Sun Q, Yu C, Hegde M, Li J, Wynn RM, Chuang DT, Hutson S, Lee B. Phenylbutyrate therapy for maple syrup urine disease. Hum Mol Genet 2011;20:631e40. 6. Mazariegos GV, Morton DH, Sindhi R, Soltys K, Nayyar N, Bond G, Shellmer D, Shneider B, Vockley J, Strauss KA. Liver transplantation for classical maple syrup urine disease: longterm follow-up in 37 patients and Comparative United Network for Organ Sharing experience. J Pediatr 2012;160:116e21.
Martı́nez-Pardo M, 7. Dı́az VM, Camarena C, de la Vega A,
pez M, Herna
ndez F, Andre
s A, Jara P. Liver Dı́az C, Lo transplantation for classical maple syrup urine disease: longterm follow-up. J Pediatr Gastroenterol Nutr 2014;59:636e9. 7 8. Yudkoff M, Daikhin Y, Nissim I, Horyn O, Luhovyy B, Lazarow A, Nissim I. Brain amino acid requirements and toxicity: the example of leucine. J Nutr 2005;135:1531Se8S. 9. Langenbeck U, Wendel U, Mench-Hoinowski A, Kuschel D, Becker K, Przyrembel H, Bremer HJ. Correlations between branched-chain amino acids and branched-chain alpha-keto acids in blood in maple syrup urine disease. Clin Chim Acta 1978;88:283e91. 10. Zinnanti WJ, Lazovic J, Griffin K, Skvorak KJ, Paul HS, Homanics GE, Bewley MC, Cheng KC, Lanoue KF, Flanagan JM. Dual mechanism of brain injury and novel treatment strategy in maple syrup urine disease. Brain 2009;132(Pt 4):903e18. 11. Sitta A, Ribas GS, Mescka CP, Barschak AG, Wajner M, Vargas CR. Neurological damage in MSUD: the role of oxidative stress. Cell Mol Neurobiol 2014;34(2):157e65. 12. Barschak AG, Sitta A, Deon M, Barden AT, Dutra-Filho CS, Wajner M, Vargas CR. Oxidative stress in plasma from maple syrup urine disease patients during treatment. Metab Brain Dis 2008;23:71e80. 13. Scaini G, Mello-Santos LM, Furlanetto CB, Jeremias IC, Mina F, Schuck PF, Ferreira GC, Kist LW, Pereira TC, Bogo MR, Streck EL. Acute and chronic administration of the branchedchain amino acids decreases nerve growth factor in rat hippocampus. Mol Neurobiol 2013;48:581e9. 14. Puffenberger EG. Genetic heritage of the Old Order Mennonites of southeastern Pennsylvania. Am J Med Genet C Semin Med Genet 2003;121C:18e31. 15. Morton DH, Strauss KA, Robinson DL, Puffenberger EG, Kelley RI. Diagnosis and treatment of maple syrup disease: a study of 36 patients. Pediatrics 2002;109:999e1008. ~ eiras DE, Bo
veda MD, Ban ~ a A, Cocho JA, 16. Couce ML, Castin
n C, Alonso-Ferna
ndez JR, Fraga JM. Iglesias AJ, Colo Evaluation and long-term follow-up of infants with inborn errors of metabolism. Mol Genet Metab 2011;104:470e5. 17. Wang YP, Qi ML, Li TT, Zhao YJ. Two novel mutations in the BCKDHB gene (R170H, Q346R) cause the classic form of maple syrup urine disease (MSUD). Gene 2012;498:112e5. 18. Yang N, Han L, Gu X, Ye J, Qiu W, Zhang H, Gong Z, Zhang Y. Analysis of gene mutations in Chinese patients with maple syrup urine disease. Mol Genet Metab 2012;106: 412e8. 19. Flaschker N, Feyen O, Fend S, Simon E, Schadewaldt P, Wendel U. Description of the mutations in 15 subjects with variant forms of maple syrup urine disease. J Inherit Metab Dis 2007;30(6):903e9. € tter J, Konstantopoulou V, Ratsch20. Simon E, Fingerhut R, Baumko Mann R, Wendel U. Maple syrup urine disease: favourable effect of early diagnosis by newborn screening on the neonatal course of the disease. J Inherit Metab Dis 2006;29:532e7. 21. Heldt K, Schwahn B, Marquardt I, Grotzke M, Wendel U. Diagnosis of MSUD by newborn screening allows early intervention without extraneous detoxification. Mol Genet Metab 2005;84(4):313e6. ~ eiras Ramos DE, Bo
vedaFonta
n MD, 22. Couce Pico ML, Castin Iglesias Rodrı́guez AJ, Cocho de Juan JA, Fraga Bermúdez JM. Advances in the diagnosis and treatment of maple syrup urine disease: experience in Galicia (Spain). An Pediatr (Barc) 2007;67:337e43. 23. Myers KA, Reeves M, Wei XC, Khan A. Cerebral edema in maple syrup urine disease despite newborn screening diagnosis and early initiation of treatment. JIMD Rep 2012;3:103e6.
ndez Sa
nchez A, Sa
nchez-Valverde 24. Dalmau Serra J, Ferna
stico y tratamiento de la Visus F. Protocolo de diagno enfermedad de jarabe de arce. In: Sanjurjo P, Couce ML, Pintos G, Ribes A, Merinero B, editors. Protocolos de diagn
ostico y tratamiento de los Errores Cong
enitos del Metabolismo AECOM. ~ a: Enar S.A.; 2007. p. 17e28. Espan Please cite this article in press as: Couce ML, et al., Evolution of maple syrup urine disease in patients diagnosed by newborn screening versus late diagnosis, European Journal of Paediatric Neurology (2015), http://dx.doi.org/10.1016/j.ejpn.2015.07.009 8 e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 5 ) 1 e8 25. Bjornson KF, Graubert CS, Buford VL, McLaughlin J. Validity of the gross motor function measure. Pediatr Phys Ther 1998;10:43e7.
mez26. Rodrı́guez-Pombo P, Navarrete R, Merinero B, Go Puertas P, Ugarte M. Mutational spectrum of maple syrup urine disease in Spain. Hum Mutat 2006;27:715. 27. Wynn RM, Davie JR, Chuang JL, Cote CD, Chuang DT. Impaired assembly of E1 decarboxylase of the branched-chain alphaketoacid dehydrogenase complex in type IA maple syrupurinedisease. J Biol Chem 1998;273:13110e8. 28. Henneke M, Flaschker N, Helbling C, Müller M, Schadewaldt P, € rtner J, Wendel U. Identification of twelve novel mutations Ga in patientswithclassic and variantforms of maple syrupurinedisease. Hum Mutat 2003;22:417. 29. Nellis MM, Danner DJ. Gene preference in maple syrup urine disease. Am J Hum Genet 2001;68:232e7. 30. Tsuruta M, Mitsubuchi H, Mardy S, Miura Y, Hayashida Y, Kinugasa A, Ishitsu T, Matsuda I, Indo Y. Molecular basis of intermittent maple syrupurinedisease: novel mutations in the E2 gene of the branched-chain alpha-ketoacid dehydrogenase complex. J Hum Genet 1998;43:91e100. 31. McConnell BB, Burkholder B, Danner DJ. Two new mutations in the human E1 beta subunit of branched chain alphaketoacid dehydrogenase associated with maple syrup urine disease. Biochim Biophys Acta 1997;1361:263e71. 32. Funchal C, Tramontina F, Quincozes dos Santos A, Fraga de Souza D, Gonçalves CA, Pessoa-Pureur R, Wajner M. Effect of 33. 34. 35. 36. 37. 38. the branched-chain alpha-keto acids accumulating in maple syrup urine disease on S100B release from glial cells. J Neurol Sci 2007;260:87e94. Muelly ER, Moore GJ, Bunce SC, Mack J, Bigler DC, Morton DH, Strauss KA. Biochemical correlates of neuropsychiatric illness in maple syrup urine disease. J Clin Invest 2013;123:1809e20. Yunus Z, Kamaludin DA, Mamat M, Choy YS, Nqu L. Clinical and biochemical profiles of maple syrup urine disease in Malaysian children. JIMD Rep 2012;5:99e107. Carecchio M, Schneider SA, Chan H, Lachmann R, Lee PJ, Murphy E, Bhatia KP. Movement disorders in adult surviving patients with maple syrup urine disease. Mov Disord 2011;26:1324e8. Fernstrom JD. Large neutral amino acids: dietary effects on brain neurochemistry and function. Amino Acids 2013;45:419e30. Strauss KA, Wardley B, Robinson D, Hendrickson C, Rider NL, Puffenberger EG, Shellmer D, Moser AB, Morton DH. Classical maple syrup urine disease and brain development: principles of management and formula design. Mol Genet Metab 2010;99:333e45. Hoffmann B, Helbling C, Schadewaldt P, Wendel U. Impact of longitudinal plasma leucine levels on the intellectual outcome in patients with classic MSUD. Pediatr Res 2006;59: 17e20. Please cite this article in press as: Couce ML, et al., Evolution of maple syrup urine disease in patients diagnosed by newborn screening versus late diagnosis, European Journal of Paediatric Neurology (2015), http://dx.doi.org/10.1016/j.ejpn.2015.07.009