The phenotypic spectrum of organic acidurias and urea cycle - - PDF document

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ORIGINAL ARTICLE

The phenotypic spectrum of organic acidurias and urea cycle

• disorders. Part 1: the initial presentation

Stefan Kölker & Angeles Garcia Cazorla & Vassili Valayannopoulos & Allan M. Lund & Alberto B. Burlina & Jolanta Sykut-Cegielska & Frits A. Wijburg & Elisa Leão Teles & Jiri Zeman & Carlo Dionisi-Vici & Ivo Barić & Daniela Karall & Persephone Augoustides-Savvopoulou & Lise Aksglaede & Jean-Baptiste Arnoux & Paula Avram & Matthias R. Baumgartner & Javier Blasco-Alonso & Brigitte Chabrol & Anupam Chakrapani & Kimberly Chapman & Elisenda Cortès i Saladelafont & Maria L. Couce & Linda de Meirleir & Dries Dobbelaere & Veronika Dvorakova & Francesca Furlan & Florian Gleich & Wanda Gradowska & Stephanie Grünewald & Anil Jalan & Johannes Häberle & Gisela Haege & Robin Lachmann & Alexander Laemmle & Eveline Langereis & Pascale de Lonlay & Diego Martinelli & Shirou Matsumoto & Chris Mühlhausen & Hélène Ogier de Baulny & Carlos Ortez & Luis Peña-Quintana & Danijela Petković Ramadža & Esmeralda Rodrigues & Sabine Scholl-Bürgi & Etienne Sokal & Christian Staufner & Marshall L. Summar & Nicholas Thompson & Roshni Vara & Inmaculada Vives Pinera & John H. Walter & Monique Williams & Peter Burgard

Received: 28 August 2014 /Revised: 21 January 2015 /Accepted: 26 January 2015 # SSIEM 2015

Abstract Background The clinical presentation of patients with organic acidurias (OAD) and urea cycle disorders (UCD) is variable; symptoms are often non-specific. Aims/methods To improve the knowledge about OAD and UCD the E-IMD consortium established a web-based patient registry. Results We registered 795 patients with OAD (n=452) and UCD (n=343), with ornithine transcarbamylase (OTC) defi- ciency (n=196), glutaric aciduria type 1 (GA1; n=150) and methylmalonic aciduria (MMA; n=149) being the most fre- quent diseases. Overall, 548 patients (69 %) were symptom-

• atic. The majority of them (n=463) presented with acute met-

abolic crisis during (n=220) or after the newborn period (n= 243) frequently demonstrating impaired consciousness, vomiting and/or muscular hypotonia. Neonatal onset of symp- toms was most frequent in argininosuccinic synthetase and lyase deficiency and carbamylphosphate 1 synthetase defi- ciency, unexpectedly low in male OTC deficiency, and least frequently in GA1 and female OTC deficiency. For patients with MMA, propionic aciduria (PA) and OTC deficiency (male and female), hyperammonemia was more severe in met- abolic crises during than after the newborn period, whereas metabolic acidosis tended to be more severe in MMA and PA patients with late onset of symptoms. Symptomatic patients without metabolic crises (n=94) often presented with a move- ment disorder, mental retardation, epilepsy and psychiatric disorders (the latter in UCD only). Conclusions The initial presentation varies widely in OAD and UCD patients. This is a challenge for rapid diagnosis and early start of treatment. Patients with a sepsis-like neona- tal crisis and those with late-onset of symptoms are both at risk

• f delayed or missed diagnosis.

Abbreviations ARG1 Arginase 1 ASL Argininosuccinate lyase ASS Argininosuccinate synthetase CPS1 Carbamylphosphate synthetase 1 E- HOD European network and registry for homocystinurias and methylation defects

Communicated by: K. Michael Gibson For a complete list of affiliations of all authors see end of this article. Electronic supplementary material The online version of this article (doi:10.1007/s10545-015-9839-3) contains supplementary material, which is available to authorized users.

• S. Kölker (*): F. Gleich: G. Haege: C. Staufner: P. Burgard

Department of General Pediatrics, Division of Inherited Metabolic Diseases, University Children’s Hospital Heidelberg, Im Neuenheimer Feld 430, D-69120 Heidelberg, Germany e-mail: Stefan.Koelker@med.uni-heidelberg.de J Inherit Metab Dis DOI 10.1007/s10545-015-9839-3

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E- IMD European registry and network for intoxication type metabolic diseases GA1 Glutaric aciduria type 1 HHH Hyperornithinemia-hyperammonemia- homocitrullinuria IVA Isovaleric aciduria MMA Methylmalonic aciduria NAGS N-acetylglutamate synthase OAD Organic aciduria OTC Ornithine transcarbamylase PA Propionic aciduria Q Quartile QoL Quality of life UCD Urea cycle disorder Introduction The clinical presentation of patients with inherited organic acidurias (OAD) and urea cycle disorders (UCD) is variable with first symptoms starting as early as the first day of life and as late as adulthood. The majority of OAD and UCD patients are thought to experience (recurrent) acute metabolic crises which are often precipitated by conditions that are likely to induce a catabolic state, or by excessive protein intake. The disease may progress from unspecific symptoms like feeding refusal and vomiting to coma, multi-organ failure and death (Ah Mew N et al 2013; Hörster et al 2009; Pena et al 2012; van der Meer et al 1994; Summar et al 2008; Zwickler et al 2014). Unlike UCD and classic OAD, untreated patients with glutaric aciduria type 1 (GA1; OMIM #231670) are at high risk to develop striatal injury often precipitated by catabolic stress during infancy (Kölker et al 2006; Strauss et al 2007). Since metabolic crises are acute life-threatening events, OAD and UCD patients require immediate and adequate metabolic emergency treatment (Baumgartner et al 2014; Chapman et al 2012; Häberle et al 2012; Sutton et al 2012). To prevent met- abolic emergencies and to reduce high mortality and morbid- ity, early diagnosis and immediate start of metabolic treatment in asymptomatic newborns is thought to improve the outcome (Enns et al 2007; Heringer et al 2010; Kölker et al 2007; Kölker et al 2011). Some individuals with OAD and UCD, however, may not develop a single episode of acute metabolic emergency during their whole life. They may remain asymp- tomatic or, more likely, may present with late-onset organ- specific symptoms that are likely to reflect chronic toxicity (Pena et al 2012; Rüegger et al 2014; Summar et al 2008). Since OAD and UCD are rare diseases and most studies have been performed in small cohorts, there is still uncertainty about the spectrum of the clinical presentation of affected individuals. Therefore, the major aim of this international study is to elucidate the clinical presentation of OAD and UCD focusing on the mani- festation of the first symptoms. Patients and methods European registry and network for intoxication type metabolic diseases (E-IMD) The European registry and network for intoxication type met- abolic diseases (E-IMD, EAHC no. 2010 12 01) is a project which has received funding from the European Union, in the framework of the Health Programme. It has been realized without industrial sponsoring. A detailed description of E- IMD will be published separately (Kölker et al 2015). Inclusion and exclusion criteria The E-IMD registry is a web-based, password-protected reg- istry (URL: https://www.eimd-registry.com). It contains comprehensive information on patients with confirmed diagnosis of an OAD, i.e. glutaric aciduria type 1 (GA1), methylmalonic aciduria (MMA; OMIM #251000, #251100, #251110, #277400, #277410), propionic aciduria (PA; OMIM #606054) and isovaleric aciduria (IVA; (OMIM #243500), or an UCD, i.e. inherited deficiency of N-acetylglutamate syn- thase (NAGS; EC 2.3.1.1; OMIM #237310), carbamylphosphate synthetase 1 (CPS1; EC 6.3.4.16; OMIM #237300), ornithine transcarbamylase (OTC, includ- ing female OTC carriers; EC 2.1.3.3; OMIM #311250), argininosuccinate synthetase (ASS; EC 6.3.4.5; OMIM #215700), argininosuccinate lyase (ASL; EC 4.3.2.1; OMIM #207900) or arginase 1 (ARG1; EC 3.5.3.1; OMIM #207800), and hyperornithinemia-hyperammonemia-homocitrullinuria (HHH; OMIM #238970) syndrome. The study was approved by the local ethic committee of the coordinating centre (i.e. University Hospital Heidelberg, application no. S-525/2010) and then was approved by all clinical partners. Patients with

• ther inherited metabolic diseases, unconfirmed suspicion of

an OAD or UCD, or with unrelated serious comorbidities were excluded. We also did not include OAD and UCD pa- tients who died before the starting date of E-IMD (i.e. 1st January 2011). Modular construction and contents of the registry The registry is constructed in a modular way, containing a set

• f common data elements for all OAD and UCD as well as

disease-specific follow-up parameters. The pre-defined algo- rithm is summarized in Suppl. Table 1.

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The modular design of the registry is helpful for compara- tive analysis of common data elements across different dis- eases, but also for deep phenotyping. It is the prerequisite to include other disease groups and thus to cluster metabolic diseases using the same IT platform. This potential has already been demonstrated by extending the disease panel to patients with homocystinurias and methylation defects (E-HOD; https://www.ehod-registry.org), and to those with inherited biogenic amine and pterin defects (iNTD; http://www.intd-

• nline.org).

Statistical analysis For descriptive statistics, SPSS (IBM SPSS Statistics 22.0) was used. Standard deviation scores (SDS) of anthropometrical data (body length, body weight, head cir- cumference) were calculated with R (R Core Team 2014) according to the LMS method (Cole 1990) and in analogy to a previous study on GA1 (Boy et al 2013). This method pro- vides normalized growth centiles. Skewed distributions of the measurements were approximated to normal distributions by power transformation. The distribution of each variable is summarized by three parameters, the Box-Cox power L, the mean M, and the coefficient of variation S. The calculation of SDS for anthropometrical parameters was based on published reference data (Cole et al 1998; Cole et al 2011). We used these reference data because they provide complete data for the whole age range examined. As most of the patients

• f our study are White European (Suppl. Table 2) and the

UK is an immigration country, these reference data ap- peared to be appropriate. Non-anthropometrical parameters were compared using randomized median difference tests programmed in R (Richter and McCann 2007). In order to ensure the stability of the probability estimates 100,000 permutations were chosen for these tests (Smucker et al 2007). The randomized median difference test was used instead of the Mann–Whitney test because distributions were skewed and unequal (Richter and McCann 2007; Hedderich and Sachs 2012). The cut-off date for statistical analysis was 22nd October 2013. Patients with HHH syndrome, NAGS deficiency and CPS1 deficiency were excluded from some statistical analyses be- cause of small group size. In addition, patients with combined MMA and homocystinuria (CblC and CblD deficiency) were excluded from the statistical analysis. For the analysis of clinical and biochemical parame- ters, we dichotomized the group of symptomatic patients by age. According to the international definition of the neonatal period, we used the term “neonatal onset” (or early onset) if first symptoms occurred within 28 days

• f life and as “late onset” if symptoms started after that

period. Results Description of the study population From 1 February 2011 to 22 October 2013, a total of 795 patients with a confirmed OAD or UCD have been registered by E-IMD centres in 19 countries (Suppl. Table 2). Most pa- tients (n=765) were born in one of these, predominantly European, countries (n=697 White European, n=32 Asian, n=24 Black, n=33 Mixed, n=9 not stated). Another 30 pa- tients were born in 20 other countries, but, subsequently, were diagnosed and followed by an E-IMD centre (Suppl. Table 2). The sample contains detailed information on basic parameters from all patients (OAD: 452, UCD: 343; boys/men: 406, girls/ women: 389), baseline visits from 788 patients, 737 regular (annual) visits, 340 emergency and 13 fatal disease course

• visits. The evaluation of the first symptoms relies on a thor-
• ugh analysis of data obtained during the baseline visit.

The frequency of individual diseases was variable (Fig. 1). The most frequent diseases were OTC deficiency (n=196; 82 male and 114 female), GA1 (n=150) and MMA (n=149), the least frequent were ARG1 deficiency (n=10), HHH syndrome (n=9) and NAGS deficiency (n=5). Median age at baseline visit was 7.4 years [quartile (Q) 1, 3.3 years; Q3, 13.8 years] in OAD and 10.1 years (Q1, 4.1 years; Q3, 18.3 years) in UCD patients, but was more variable in single diseases (Fig. 2). The OAD sample contained 64 (14 % of OAD patients) and the UCD sample 87 adult (i.e. ≥ 18 years) patients (25 % of UCD patients) due to a high proportion of female OTC carriers in the latter group (n=82). Median age at diagnosis was 30 days (Q1, 8 days; Q3, 360 days) for OAD patients and 362 days (Q1, 8 days; Q3, 1824 days) for UCD patients (Fig. 3), but showed high variability. The age at diagnosis was unexpectedly high in male OTC patients (median, 390 days) suggesting that some patients with early-onset OTC deficiency might have been missed because of fatal neonatal crisis or might have died before the cut-off date for inclusion (i.e. 1 January 2011). Patients with late onset

• f symptoms showed more often and more pronounced

diagnostic delay than those with neonatal onset of symp- toms (Table 1). Without appropriate metabolic treatment it is likely to assume that in both groups an unknown num- ber of patients died undiagnosed. The majority of registered patients (total: 513 patients, OAD: 275 patients, UCD: 238 patients) were diagnosed after the onset of first symptoms, whereas the remaining patients were identified neonatally due to newborn screening (total: 168 patients, OAD: 137 patients, UCD: 31 patients) or met- abolic cascade testing in high-risk families with a previous- ly known index patient (total: 88 patients, OAD: 29 pa- tients, UCD: 59 patients). Six patients (OAD: two patients, UCD: four patients) were diagnosed prenatally. In 20 pa- tients, the mode of diagnosis was not reported. A detailed

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evaluation of the diagnostic algorithm will be published separately. The majority of registered patients were symptomatic, whereas about one-fourth remained asymptomatic during the study period (Table 2). In the OAD group, the majority

• f asymptomatic individuals was found in the newborn

screening group (80 of 137 neonatally screened OAD pa- tients, in particular those having GA1 or IVA). In the UCD group, most asymptomatic individuals were found in the group of female OTC carriers (30 of 114 patients), many

• f whom (n=16) were diagnosed after they had given birth

to a symptomatic newborn with OTC deficiency (13 male, three female). Pregnancy, delivery and anthropometrical parameters at birth Mothers might be affected by the metabolic disease of their unborn child. This has for instance been shown for long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (Gutiérrez Junquera et al 2009; Strauss et al 1999). In general, the fre- quency of reported maternal health problems during pregnan- cies with children having OAD (n=89) and UCD (n=40) was

• Fig. 2 Age at baseline visit in patients with OAD (a), UCD except

female OTC (b), and female OTC (c). Circles, outliers; asterisks, extreme values; height of box: interquartile range; top end of box: Q3; bottom end of box: Q1; bold line inside of box: median; top/bottom whiskers: highest/lowest case within 1.5 times interquartile range;

• utliers: distance to Q1 or Q3 respectively≥1.5 and≤3 times

interquartile range; extreme values: distance to Q1 or Q3 respectively> 3 times interquartile range

• Fig. 1 Frequency of OAD and

UCD in the E-IMD sample. Abbreviations: ARG1, arginase 1; ASL, argininosuccinate lyase; ASS, argininosuccinate synthetase; Cbl, cobalamin; CPS1, carbamylphosphate synthetase 1; D, deficiency; GA1, glutaric aciduria type 1; HHH syndr., hyperornithinemia- hyperammonemia- homocitrullinuria syndrome; IVA, isovaleric aciduria; MMA, methylmalonic acidurias; NAGS, N-acetylglutamate synthase; OAD, organic acidurias; OTC,

• rnithine transcarbamylase;

syndr, syndrome; UCD, urea cycle disorder J Inherit Metab Dis

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comparable to the normal population. Most frequently report- ed health problems were gestational diabetes (OAD, 25 % of pregnancies with reported maternal health problems: MMA: n=4, PA: n=6, IVA: n=6, GA1: n=6; UCD, 15 %: ASS: n=1, ASL: n=1, male OTC: n=2, female OTC: n=2) and arterial hypertension (OAD, 9 % of pregnancies with reported mater- nal health problems: MMA: n=5, PA: n=1, GA1: n=2; UCD, 20 %: ASS: n=1, ASL: n=1, male OTC: n=3, female OTC: n=3). Considering all pregnancies, however, the overall fre- quency of gestational diabetes in OAD pregnancies (5.5 %) and that of arterial hypertension in UCD pregnancies (3 %) was similar to that found in pregnancies in general, i.e. 2-10 % for gestational diabetes (Gabbe et al 2012) and 4.7 % for arterial hypertension (Liu et al 2014). Other medical problems were only reported in single cases for UCD [hepatic disease (ASL deficiency), renal disease (ASS deficiency)] and OAD [renal disease (GA1), psychiatric disease (IVA), pre-eclampsia (PA, IVA), eclampsia (GA1), and epilepsy (PA)]. Cardiac problems were not reported. Next, we studied whether there is evidence for fetal disease

• manifestation. We did not find an increased frequency of pre-

term infancy. Median gestational age was 40 weeks for both OAD (Q1, 38 weeks; Q3, 40 weeks) and UCD patients (Q1, 38 weeks; Q3, 40 weeks). Postnatal problems were common in OAD (n=111) and UCD (n=72) patients, with feeding problems (OAD: 20 %, UCD: 14 %) and hyperbilirubinemia (OAD: 9 %, UCD: 7 %) being most frequently reported. Some

• f these problems (e.g. feeding problems) most likely reflect

the beginning of a neonatal metabolic crisis, whereas others (e.g. hyperbilirubinemia) are found in the same range as in the general population. Anthropometrical parameters were mostly in the normal range, however, newborns with MMA showed significantly lower birth weights [t(df=645)=−6.19, p< 0.001] and GA1 patients [t(df=135.6)=5.40, p<0.001] were more often macrocephalic than newborns with other OAD and UCD (Table 3; Suppl. Table 3). In summary, except for pa- tients with GA1 and MMA our analysis did not reveal evi- dence of intrauterine disease manifestation or of increased maternal health problems during pregnancies with OAD and UCD individuals. Acute metabolic crisis — clinical presentation and metabolic derangement A metabolic crisis is an acute life-threatening event that can be precipitated by catabolism, inadequately high or low protein intake, and other causes (e.g. menstruation, postnatal uterine involution and valproate precipitating hyperammonemia). In its most severe form it already manifests during the neonatal period, usually after a symptom-free interval of a few days and clinically resembling neonatal sepsis. However, metabolic cri- ses may manifest at any age. We therefore studied the propor- tion and age at onset as well as the severity of the clinical presentation and biochemical derangement of patients in the E-IMD cohort presenting with metabolic crisis as initial symptom. Among the 548 symptomatic patients (OAD: 306, UCD: 242), 412 patients [classic OAD (i.e. without GA1): 211,

• Fig. 3 Age at diagnosis in patients with OAD (a), UCD except female

OTC (b), and female OTC (c). Median age of diagnosis by disease (in days): MMA (30), PA (16), IVA (13), GA1 (270), NAGS deficiency (7), CPS1 deficiency (194), ASS deficiency (7), ASL deficiency (15), ARG1 deficiency (39), HHH syndrome (570), male OTC deficiency (390), female OTC deficiency (1094). Circles, outliers; asterisks, extreme values; height of box: interquartile range; top end of box: Q3; bottom end of box: Q1; bold line inside of box: median; top/bottom whiskers: highest/lowest case within 1.5 times interquartile range; outliers: distance to Q1 or Q3 respectively≥1.5 and≤3 times interquartile range; extreme values: distance to Q1 or Q3 respectively>3 times interquartile range J Inherit Metab Dis

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UCD: 201] presented with an acute metabolic crisis, and 51 GA1 patients with an encephalopathic crisis as initial clinical

• manifestation. In 220 patients, acute metabolic crises occurred

during the newborn period [median age at start of first symp- toms (Q1; Q3): 3 days (Q1, 2 days; Q3, 7 days)], whereas 243 patients had a metabolic crisis or encephalopathic crisis after the newborn period [median age at start of first symptoms (range): 390 days (Q1, 196 days; Q3, 730 days)]. The propor- tion of early- versus late-onset metabolic crisis varied in single OAD and UCD (Fig. 4; Suppl. Table 4). A high frequency of neonatal crises was found in ASS, CPS1, ASL and NAGS deficiency, PA, IVA and MMA, an intermediate frequency for male OTC deficiency and HHH syndrome, and a low frequency for female OTC deficiency, GA1 and ARG1 defi- ciency (Table 2). Impaired consciousness (n=283 patients, i.e. 52 % of symptomatic patients), vomiting (n=203 patients, 37 %), and muscular hypotonia (n=177 patients, 32 %) were most often reported (Table 4). Evaluation of basic metabolic parameters showed a broad overlap. The most robust distinguishing pattern be- tween patients with classic OAD (PA, MMA, IVA) and UCD (CPS1, OTC, ASS and ASL deficiency) was normoglutaminemic hyperammonemia in OAD and hyperglutaminemic hyperammonemia in UCD, in particu- lar in OAD and UCD patients with early-onset metabolic crisis (Tables 5 and 6; Suppl. Tables 5 and 6). In the OAD group, we found higher plasma ammonia concentra- tions in early-onset MMA and PA patients than in the late-onset group, whereas the plasma glutamine concentra- tion was lower in early-onset MMA and — less pro- nounced — early-onset IVA patients than in the late-

• nset group. Interestingly, late-onset MMA and PA pa-

tients showed a stronger decrease in standard bicarbonate and — less pronounced — in pCO2 and pH (PA only) than those with early-onset of symptoms (Table 5; Suppl. Table 5). In the UCD group, early-onset male OTC

Table 1 Diagnostic delay in patients diagnosed after the manifestation of first symptoms Asymptomatic patients who were diagnosed while being asymptomatic (by newborn screening, high-risk family screening, prenatal testing) or symptomatic patients with incomplete clinical data (OAD: 40; UCD: 34) were excluded from this analysis Abbreviations: D, deficiency; DoL, day of life; EO, early onset

• f symptoms (during the newborn

period, i.e. ≤28 days); f, female; LO, late onset of symptoms (after the newborn period, i.e. >28 days); m, male; syndr., syndrome Patients per

• nset type

Time to diagnosis after onset of first symptoms 1-7 days 8-31 days 32-365 days ≥365 days (n) (n) (n) (n) (n) OAD EO 80 51 17 8 4 LO 148 79 11 32 26 UCD EO 68 57 2 6 3 LO 129 73 9 25 22 MMA EO 38 28 6 2 2 LO 48 28 4 12 4 PA EO 27 19 6 2 LO 25 16 5 2 2 IVA EO 11 4 4 1 2 LO 12 4 2 6 GA-I EO 4 1 3 LO 63 31 2 16 14 NAGS-D EO 1 1 LO 1 1 CPS1-D EO 7 3 1 3 LO 5 2 3 OTC-D (m) EO 16 16 LO 39 25 4 5 5 OTC-D (f) EO 3 2 1 LO 52 28 4 10 10 ASS-D EO 28 24 1 3 LO 12 8 1 3 ASL-D EO 13 11 1 1 LO 12 6 3 3 ARG1-D EO LO 3 2 1 HHH syndr. EO LO 5 3 2 J Inherit Metab Dis

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patients had a significantly higher concentration of ammo- nia, glutamine, ornithine, orotic acid and lactate, and a lower concentration of standard bicarbonate and citrulline than late-onset patients. Similarly, symptomatic female OTC carriers who had a neonatal metabolic crisis had a higher ammonia and tendency towards higher lactate and lower pH than those with late onset of symptoms. Although early-onset patients with CPS1, ASS and ASL deficiency also seemed to have higher plasma ammonia and glutamine concentrations than late-onset patients, sta- tistical analysis did not confirm significance (Table 6;

• Suppl. Table 6).

As expected, none of the patients with GA1 presented with a ‘classic’ neonatal metabolic crisis.

Table 2 Frequency of symptomatic and asymptomatic UCD and OAD patients by disease Abbreviations: D, deficiency; EO, early onset (i.e. patients with a neonatal crisis/≤28 days); f, female; LO, late onset (i.e. patients with crisis after the newborn period/>28 days); LO w/o AC, late onset without acute crisis (i.e. symptomatic patients without an acute metabolic crisis); m, male; syndr., syndrome Disease group Disease name Patients (n) Symptomatic (n) Asymptomatic (n) All EO LO LO w/o AC OAD MMA 149 116 56 46 14 33 PA 101 82 53 27 2 19 IVA 52 31 17 12 2 21 GA1 150 77 3 48 26 73 Total (OAD) 452 306 129 133 44 146 UCD NAGS-D 5 4 3 1 1 CPS1-D 18 16 11 3 2 2 OTC-D (m) 82 61 19 38 4 21 OTC-D (f) 114 70 4 49 17 44 ASS-D 61 48 33 8 7 13 ASL-D 44 32 18 5 9 12 ARG1-D 10 4 1 3 6 HHH syndr. 9 7 2 3 2 2 Total (UCD) 343 242 91 110 41 101 OAD+UCD Total 795 548 220 243 85 247 Table 3 Gestational age and anthropometrical parameters at birth Disease name Patients per disease Gestational age Body weight Supine length Head circumference n n Weeks (Median) n SDS (Median) n SDS (Median) n SDS (Median) MMA 149 126 40 127 −0.81 107 −0.50 87 PA 101 84 39 91 −0.29 81 −0.15 71 0.44 IVA 52 47 39 45 −0.04 39 31 GA-I 150 131 40 131 −0.28 114 102 0.88 NAGS-D 5 3 38 3 −1.31 3 −1 3 CPS1-D 18 13 40 14 −0.10 13 13 0.37 OTC-D (f) 114 71 40 70 −0.29 55 43 OTC-D (m) 82 63 39 65 −0.36 56 −0.33 49 ASS-D 61 52 40 51 −0.24 44 −0.13 42 −0.44 ASL-D 44 38 39 36 −0.47 31 −0.43 29 ARG1-D 10 8 39.5 8 −0.36 6 −0.79 6 0.11 HHH syndr. 9 7 40 6 0.18 5 0.43 4 0.75 For descriptive statistical information including median, mean, minimum, maximum, interquartile range see Suppl. Table 3 Abbreviations: D, deficiency; f, female; m, male; SDS, standard deviation score; syndr., syndrome Bold types indicate statistically significant anthropometric parameters, i.e. low birth weight in MMA patients and increased head circumference in GA1 patients J Inherit Metab Dis

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Other signs and symptoms leading to the diagnosis Among the 328 patients with late onset of symptoms, 243 patients presented with an acute metabolic crisis, whereas 85 patients did not develop a metabolic crisis. The majority of patients with late-onset metabolic crises, however, had signs and symptoms that preceded the crisis, but that were only retrospectively recognized as initial symptoms. In late-onset OAD patients, movement disorders (n= 34), mental retardation (n=20) and epilepsy (n=15) were most frequently found. Similarly, late-onset UCD patients most often presented with mental retardation (n=18), movement disorder (n=12), epilepsy (n=11) and psychiatric disorders (n=9) (Table 7). In GA1, 12

• f 74 late-onset patients (16 %) showed epilepsy as

initial clinical presentation which is above the frequency for epilepsy in the general population. Furthermore, a dystonic movement disorder without preceding encepha- lopathic crisis was reported in 29 of 74 late-onset GA1 patients (39 %) highlighting a high frequency of insidious-onset dystonia. In late-onset female but not in male OTC patients, liver disease ranging from elevat- ed liver enzymes to acute liver failure was observed in 17 of 64 patients (27 %) as the initial presentation. Notably, seven of 14 patients with late-onset ASL defi- ciency (50 %) presented with mental retardation. Six of them never had a documented metabolic crisis. Discussion The aim of this study was to describe and evaluate the initial clinical presentation of patients with OAD (n= 452) and UCD (n=343). The proportion of single UCD was in a similar range as described in samples from the US (Batshaw et al 2014; Summar et al 2013), Japan (Kido et al 2012) and France (Nassogne et al 2005). Since newborn screening programmes for OAD and UCD have so far been established in a few European countries (Burgard et al 2012; Loeber et al 2012) and symptomatic patients might be missed for various reasons, it is quite likely that the E-IMD dataset is skewed and under-represents patients with a severe phenotype to some extent. In line with this, we previously demonstrated that estimated minimum prev- alences for OAD and UCD varied in different countries (Kölker et al 2015). Furthermore, the geographic dis- tribution of rare diseases is variable and relies on ge- netic differences in populations such as localized foun- der effects. This should be taken into consideration while discussing this study. The major results of this study are: (1) anthropomet- ric parameters at birth in MMA (low birth weight) and GA1 patients (macrocephaly) suggest fetal disease man- ifestation; (2) the majority of symptomatic patients pre- sented with an acute metabolic (all except GA1) or acute encephalopathic crises (GA1); (3) an unexpectedly

• Fig. 4 Age at onset of first clinical symptoms in patients with OAD

(a), UCD except female OTC (b) and female OTC carriers (c). Patients with NAGS and HHH deficiency were omitted from this analysis due to small numbers in these groups. Median age (Q1;Q3) at onset of first symptoms by disease (in days): MMA: 21 (3;210), PA: 14 (4;90), IVA: 8 (5;465), GA1: 300: (145;428), CPS1 deficiency: 4 (3;26), ASS deficiency: 3 (2;18), ASL deficiency: 3(2;10), ARG1 deficiency: 2190 (34;—), male OTC deficiency: 315 (7;1283), female OTC deficiency: 690 (363;1345). Circles, outliers; asterisks, extreme values; height of box: interquartile range; top end of box: Q3; bottom end of box: Q1; bold line inside of box: median; top/bottom whiskers: highest/lowest case within 1.5 times interquartile range; outliers: distance to Q1 or Q3 respectively≥1.5 and≤3 times interquartile range; extreme values: distance to Q1 or Q3 respectively>3 times interquartile range J Inherit Metab Dis

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high number of patients, in particular those with UCD, presented with metabolic crises or other symptoms after the newborn period; and (4) in early-onset MMA, PA and OTC deficiency (male and female), peak ammonia concentrations were higher than in the late-onset group, whereas late-onset MMA and PA patients tended to have a more pronounced decrease in standard bicarbonate. When does the disease start? Accumulation of toxic metabolites in fetuses with OAD and UCD is usually thought to be maternally prevented and, therefore, first symptoms should not occur before the newborn period. In contrast, intrauterine disease manifes- tation is found in metabolic diseases affecting energy me- tabolism (DeBrosse et al 2012; Olsen et al 2003, Whitfield et al 1996). In our cohort, low birth weight in MMA patients and increased head circumference in GA1 patients were the sole indicators of potentially intrauterine disease manifestation. There is evidence of secondary mitochondrial dysfunc- tion in MMA patients which can be induced acutely by inhibition of pyruvate dehydrogenase complex and tricar- boxylic acid cycle enzymes by propionyl-CoA, 2- methylcitric acid and methylmalonic acid, and chronically by reduced activity of complexes III and IV of the respi- ratory chain (Chandler et al 2009; De Keyzer et al 2009;

Table 4 Clinical presentation during the initial metabolic crisis Disease name Patients per

• nset type

Impaired consciousness Vomiting Muscular hypotonia Sepsis-like appearance Seizures Hyper- excitability Odour Others n n Days (Median) n Days (Median) n Days (Median) n Days (Median) n Days (Median) n Days (Median) n Days (Median) n Days (Median)

• a. OAD patients

MMA EO 56 37 3 24 3 28 4 19 3 7 2 6 4 2 4 37 3 LO 46 29 270 32 210 20 195 6 183 6 165 6 93 1 180 22 240 PA EO 53 36 7 24 12 31 7 18 4 7 14 2 2 2 15 31 4 LO 27 24 195 19 180 13 150 3 240 7 180 0 n/a n/a 12 152 IVA EO 17 9 7 4 5 10 7 3 2 1 4 6 6 10 4 11 5 LO 12 3 810 9 570 4 387 2 1044 n/a 0 n/a 2 1800 5 390

• b. UCD patients

NAGS-D EO 2 1 6 1 6 1 2 n/a n/a 0 n/a n/a 1 2 LO 1 1 3650 n/a n/a 1 3650 n/a 0 n/a n/a n/a CPS1-D EO 11 7 3 3 2 3 4 3 2 4 4 2 6 n/a 8 3 LO 3 3 1200 1 1200 1 84 1 6570 n/a 1 6570 1 1200 n/a OTC-D (f) EO 4 3 4 2 6 1 3 3 4 n/a 0 n/a n/a 2 3 LO 49 33 730 29 690 12 525 2 1230 8 1043 3 1230 n/a 22 600 OTC-D (m) EO 19 13 3 6 7 8 3 12 2 10 2 2 2 n/a 8 3 LO 38 31 725 27 510 11 365 2 2538 9 360 7 810 1 2555 10 378 ASS-D EO 33 23 3 7 2 21 3 14 3 13 3 4 4 n/a 17 3 LO 8 6 360 5 480 2 103 n/a n/a 1 480 n/a 2 540 ASL-D EO 18 14 3 4 5 5 3 4 3 9 3 3 2 n/a 9 2 LO 5 4 740 3 750 3 180 1 730 2 2450 0 n/a n/a 2 2138 ARG1-D EO 1 1 n/a n/a n/a n/a 0 n/a n/a n/a LO 3 1 2190 1 34 1 2190 2 1112 1 2190 1 2190 n/a 1 2190 HHH-D EO 2 2 4 n/a 1 2 1 2 1 2 1 2 n/a 1 6 LO 3 2 540 2 540 1 180 n/a n/a 1 450 n/a 2 315 Patients with GA1 are excluded from this table, since they present with an encephalopathic crisis but not with a classic metabolic crisis. For descriptive statistical information including median, mean, minimum, maximum, interquartile range see Suppl. Table 4a Abbreviations: EO, early onset of symptoms (during the newborn period/≤28 days); LO, late onset of symptoms (after the newborn period/>28 days); n/a, not applicable For descriptive statistical information including median, mean, minimum, maximum, interquartile range see Suppl. Table 4b. Abbreviations: D, deficiency; EO, early onset of symptoms (during the newborn period); f, female; LO, late onset of symptoms (after the newborn period); m, male; n/a, not applicable J Inherit Metab Dis

SLIDE 10

Okun et al 2002; Schwab et al 2006). It is therefore tempting to speculate that low birth weight in MMA pa- tients might reflect antenatally impaired energy metabo- lism (Tavares et al 2013). Since PA and MMA share bio- chemical and pathomechanistic similarities, it remains to be elucidated why birth weights of these patients differed in our study. Whereas low birth weight in MMA patients has not yet been described in a larger population, macrocephaly is a well-known finding in GA1 patients. It is found in approximately 75 % of GA1 patients during the new- born period and infancy, whereas the head circumfer- ence often tends to normalize with increasing age (Boy et al 2013; Kölker et al 2006). MRI studies have unraveled an immature gyral pattern, delayed white mat- ter development and temporal hypoplasia in newborns with GA1 (Harting et al 2009). The underlying mecha- nism of macrocephaly and delayed fetal brain develop- ment is not known. Disturbed cerebral hemodynamics with secondarily expanded cerebrospinal fluid volume has been suggested as potential mechanism (Strauss et al 2010). Furthermore, cerebral accumulation of glutaric and 3-hydroxyglutaric acid due to entrapment by the blood–brain barrier, which has a low efflux ca- pacity for dicarboxylic acids, might already play a role in utero since the blood–brain barrier differentiates and continuously matures during fetal development (Engelhardt and Liebner 2014; Sauer et al 2006; Sauer et al 2010). Accumulating glutaric and 3- hydroxyglutaric acid are putative neurotoxins and are thought to be relevant for inducing neurological symp- toms in infancy (Kölker et al 2002; Kölker et al 2013; Lamp et al 2011; Sauer et al 2005), whereas it is not known whether they already affect the fetal brain. Onset of first symptoms during and after the newborn period The majority of symptomatic patients with OAD (262 of 306 symptomatic patients) and UCD (201 of 242 symp- tomatic patients) presented with an acute metabolic cri- sis as early as the first day of life or as late as adult-

• hood. Neonatal onset of symptoms is thought to reflect

low residual enzyme activity and severe disease course in classic OAD and UCD (Enns et al 2007; Hörster et al 2009; Nassogne et al 2005; Vockley and Ensenauer 2006). For classic OAD, we demonstrated a similar frequency of patients presenting neonatally (60 % of 211 with a crisis; Suppl. Table 3) compared to previous studies (Dionisi-Vici et al 2006; Grünert et al 2012; Grünert et al 2013; Hörster et al 2009; Leonard et al 2003). We also confirmed that most pa- tients with GA1 presented with irreversible neurological symptoms after the newborn period (Kölker et al 2006). Unexpectedly, however, only 91 of 201 UCD patients (45 %) with a metabolic crisis presented during the newborn period. Interestingly, a recent study from Japan showed similar results (Kido et al 2012), whereas a previous study in a US sample showed an even lower frequency (Suppl. Table 7). Since UCD patients presenting with a neonatal meta- bolic crisis have a high mortality (Enns et al 2007; Nassogne et al 2005; Summar et al 2008), we assume that the low proportion of UCD patients with a neonatal met- abolic crisis in general and that of male OTC patients in

Table 5 Biochemical pattern of acute metabolic crisis in OAD patients Disease name Patients per pH pCO2 Standard Lactate (p) Glucose (s) Ammonia (p) Glutamine (p)

• nset type

bicarbonate n Onset n — n kPa n mmol/L n mmol/L n mmol/L n μmol/L n μmol/L (Median) (Median) (Median) (Median) (Median) (Median) Median MMA 56 EO 40 7.27 37 2.77 (*) 28 13.4 * 33 2.6 27 5.2 42 323 ** 19 407 * 46 LO 30 7.18 23 2 21 8.2 16 3 22 4.5 24 117 7 705 PA 53 EO 34 7.31 * 27 3.33 (*) 26 17.4 (*) 25 2.5 23 5 43 444 * 17 466 27 LO 19 7.2 11 2.47 14 9.2 14 2.1 13 3.5 21 142 8 425 IVA 17 EO 12 7.4 (*) 11 3.51 10 20 10 1.6 8 5.2 13 239 5 371 (*) 12 LO 5 7.32 4 3.55 3 16 1 0.7 5 5.22 6 70 2 588 For descriptive statistical information including median, mean, minimum, maximum, interquartile range see Suppl. Table 5

( *) 0.1>p≥0.05, *p<0.05 vs LO of the same disease (unadjusted p-value for explorative analysis), **p<0.00146 vs LO of the same disease (randomized

median difference test with Sidak-adjusted p-value for multiple comparison of medians with 100,000 permutations). Patients with GA1 did not show a metabolic crisis and thus were not included in this table. Since not all metabolic parameters have been reported for all patients, the sample size by disease and group is shown as range Abbreviations: EO, early onset (metabolic crisis during newborn period/≤28 days); LO, late onset (metabolic crisis after the newborn period/>28 days); p, plasma; s, serum J Inherit Metab Dis

SLIDE 11

Table 6 Biochemical pattern of acute metabolic crisis in UCD patients Disease name Patients per

• nset type

pH pCO2 Standard bicarbonate Lactate (p) Glucose (s) Ammonia (p) n n — (Median) n kPa (Median) n mmol/L (Median) n mmol/L (Median) n mmol/L (Median) n μmol/L (Median) CPS1-D EO 11 4 7.51 (*) 3 2.9 4 20 2 5.9 3 9.8 7 600 LO 3 2 7.43 1 4.1 1 15 2 1.4 2 5 3 128 OTC-D (f) EO 4 2 7.31 (*) 2 4.9 1 18 2 6.6 (*) n/a 3 782 * LO 49 25 7.44 22 3.8 21 21.9 22 2.5 24 5.2 38 253 OTC-D (m) EO 19 13 7.35 12 4.3 8 18 * 14 3.8 * 11 5.5 18 2032 ** LO 38 20 7.4 13 4 16 23 12 1.5 15 0.6 28 179 ASS-D EO 33 23 7.4 20 4.3 17 18 * 23 4 21 5.6 30 656 LO 8 3 7.36 2 5.2 3 23 1 0.8 2 4.2 5 220 ASL-D EO 18 13 7.4 9 3.8 9 19 9 3.8 8 5.4 12 687 LO 5 2 7.36 1 2.5 3 22 1 3.1 3 6.7 4 447 Disease name Glutamine (p) Citrulline (p) Arginine (p) Ornithine (p) Arginino-succinate (p) Orotic acid (u) n μmol/L (Median) n μmol/L (Median) n μmol/L (Median) n μmol/L (Median) n μmol/L (Median) n mmol/mol creatinine (Median) CPS1-D 7 1105 8 1 6 18 5 49 1 4 3 n/a n/a 1 58 n/a n/a n/a OTC-D (f) n/a n/a n/a n/a n/a n/a 23 895 24 12 20 22 18 35 2 5.5 26 229 OTC-D (m) 15 1865 ** 13 0 * 11 23 11 103 * 3 8 815 * 23 965 21 12 21 28 21 23 2 14 228 ASS-D 21 1358 24 2058 16 36 17 60 5 13 552 5 758 5 1192 3 23 3 21 n/a 2 68 ASL-D 9 1922 9 256 8 26 7 37 7 240 3 72 2 970 3 96 3 61 3 52 3 295 4 48 For descriptive statistical information including median, mean, minimum, maximum, interquartile range see Suppl. Table 6 (*)0.1>p≥0.05, *p<0.05 vs LO of the same disease (unadjusted p-value for explorative analysis), **p<0.00081 vs LO of the same disease (randomized median difference test with Sidak-adjusted p-value for multiple comparison of medians with 100,000 permutations). Patients with NAGS, ARG1 and HHH deficiency were omitted due to small numbers. Since not all metabolic parameters have been reported for all patients, the sample size by disease and group is shown as range Abbreviations: D, deficiency; EO, early onset (metabolic crisis during newborn period/≤28 days); f, female; LO, late onset (metabolic crisis after the newborn period/>28 days); m, male; n/a, not applicable; p, plasma; s, serum; u, urine J Inherit Metab Dis

SLIDE 12

Table 7 Initial clinical presentation of patients with late-onset disease manifestation Patients (n) Feeding problems (n) Hepatic manifestation (n) Epilepsy (n) Movement disorders (n) Mental retardation (n) Psychiatric disorder (n) Renal manifestation (n) Abnormal WBC (n) Cardiac manifestation (n) Others (n) MMA AC 46 14 1 2 3 2 1 10 w/o 14 3 1 2 1 2 PA AC 27 11 1 2 5 1 2 9 w/o 2 1 1 IVA AC 12 2 1 5 w/o 2 1 1 1 GA1 AC 48 9 7 17 6 1 18 w/o 26 2 5 12 4 15 NAGS-D AC 1 1 w/o CPS1-D AC 3 3 1 1 3 w/o 2 1 1 2 OTC-D (m) AC 38 9 2 3 4 2 2 9 w/o 4 4 1 1 OTC-D (f) AC 49 9 12 3 2 1 4 1 17 w/o 17 8 5 2 1 1 7 ASS-D AC 8 3 3 2 w/o 7 4 1 1 2 2 2 ASL-D AC 5 1 1 1 1 3 w/o 9 1 2 2 6 2 ARG1-D AC 3 1 1 1 1 w/o HHH syndr. AC 3 2 2 1 1 w/o 2 1 1 Abbreviations: AC, acute metabolic crisis after the newborn period/>28 days; f, female; m, male; syndr., syndrome; WBC, whole blood cell count; w/o, late disease onset without metabolic crisis J Inherit Metab Dis

SLIDE 13

particular reflects that many UCD patients might have died undiagnosed and thus are missing in the E-IMD sam-

• ple. This notion is supported by a higher frequency of

neonatal crises in two other studies, a monocentric study from France (Nassogne et al 2005) and an ongoing trans- national epidemiological study in Germany, Austria and Switzerland (Nettesheim et al 2013). Whereas the onset of an acute metabolic crisis was the common presentation in OAD and UCD patients with neonatal onset, 85 of 328 (i.e. 26 %) of patients who presented after the newborn period never developed an acute crisis. Most frequently these patients developed neurological symptoms such as movement disorders, mental retardation and epilepsy, and psychiatric disease (UCD only). These symptoms have a broad spectrum of differential diagnoses, and thus the correct diagnosis could be delayed or even missed. Notably, we observed a high frequency of epilepsy and insidious-onset dystonia in late-onset GA1 patients (Table 7). So far epilepsy has sporadically been reported as the initial presentation in GA1 (McClelland et al 2009), and the frequency of insidious-onset dystonia was lower in two international studies (Hoffmann et al 1996; Kölker et al 2006). Besides neurological manifestation, gastrointestinal symp- toms, in particular feeding problems, were often observed in late-onset patients. Interestingly, hepatomegaly, (transiently) elevated liver enzymes and acute liver failure were frequently found in female OTC carriers (Table 7). This is an important result since knowing about the he- patic manifestation in female OTC deficiency could be the key to identify these patients before severe decompensa- tion (Gallagher et al 2014; Mustafa and Clarke 2006; Teufel et al 2011). In the follow-up of treated patients, however, hepatomegaly and liver dysfunction may alter- natively indicate malnutrition because of excessive dietary treatment. The age at onset in classic OAD and UCD patients is thought to correlate with their residual enzyme activity and the severity of the concomitant metabolic derange-

• ment. In the E-IMD sample, this assumption was con-

firmed for patients with early-onset MMA, PA and OTC deficiency (male and female) whose peak plasma ammo- nia concentration during the initial metabolic crisis was significantly higher than those of late-onset patients (Tables 5 and 6; Suppl. Tables 5 and 6). These results are in line with previous studies (Enns et al 2007; Summar et al 2008; Zwickler et al 2012; Zwickler et al 2014). In contrast, late-onset MMA and PA patients tended to have a more pronounced decrease in standard bicarbonate than early-onset patients (Table 5; Suppl. Table 5). This may reflect age-dependent physiologic changes, e.g. tubular function, or differences in the affin- ity of mitochondrial enzymes to toxic metabolites. Therefore, these metabolic discrepancies should be care- fully evaluated, in particular with regard to their predic- tive and prognostic value. Rapid diagnosis in OAD and UCD: still a challenge Once an OAD or UCD is suspected, the diagnosis can usually be rapidly confirmed by a specialized metabolic

• laboratory. In single patients with intermittently normal

metabolic parameters such as female OTC carriers and GA1 patients with a low excreter phenotype, however, the diagnostic process might be time-consuming. The major obstacle in identifying OAD and UCD patients rapidly and to start metabolic treatment without delay is the initiation of the diagnostic process itself (Rüegger et al 2014). The clinical presentation of OAD and UCD patients is non-specific including sepsis-like acute met- abolic crises in the newborn period and feeding prob- lems, mental retardation, epilepsy and movement disor- ders in late-onset patients. To improve the prognosis of OAD and UCD patients, it is important to further in- crease the awareness of physicians about these rare dis- eases and to establish programmes that allow early identification of affected individuals with OAD and

• UCD. GA1 (n=10 countries) and IVA (n=9 countries)

are increasingly included in newborn screening programmes in Europe, and some countries also screen for MMA (isolated forms, n=1; combined forms, n=7), PA (n = 7), ASL (n = 6) and ASS deficiency (n = 5) (Burgard et al 2012; Loeber et al 2012). Newborn screening for some UCD (NAGS and CPS1 deficiency) is technically challenging, and screening of female OTC carriers results in a diagnostic and ethical dilem-

• ma. Except for single diseases such as GA1, there is

still uncertainty whether patients with OAD and UCD will benefit from newborn screening (Kölker et al 2007; Heringer et al 2010). Therefore, more work is required to understand whether newborn screening for MMA, PA and UCD improves early identification of patients, pre- vents the manifestation of acute metabolic crises and alters their long-term outcome.

Acknowledgments We are indebted to all patients and their families who have been willing to contribute to this study, to share their experience

• n living with a rare disease, and for their trust, and we thank all col-

leagues very much for their contribution to the project. We are grateful for fruitful collaboration with the following clinical partners, patient support groups and industrial partners (in alphabetical order of countries): Lut de Baere, Nathalie Stroobant (Belgische Organisatie voor Kinderen en Volwassenen met een Stofwisselingsziekte VZW [BOKS], Belgium), Nela Carić (Hrvatska udruga za rijetke bolesti, Croatia), Tomas Honzik (Charles University and General University of Prague, First Faculty of Medicine, Prague, Czech Republic), Annika and Kennet Rovsing (PND - Protein Nedbrydnings Defekt Foreningen, Denmark), Samantha Parker J Inherit Metab Dis

SLIDE 14

(Orphan Europe SARL, France), EURORDIS, European Organisation for Rare Disease (France), Eric Bauchart (Hôpital Necker-Enfants Malades, Assistance Publique-Hôpitaux de Paris, Reference Center for Inherited Metabolic Disease, Necker-Enfants Malades University Hospi- tal and IMAGINE Institute, Paris, France), Markus Ott, Beate Szczerbak (Nutricia Metabolics GmbH, Germany), Hubertus von Voss, Raimund Schmid (Kindernetzwerk e.V., Germany), Mandy Kretschmer (Glutarazidurie e.V., Germany), Reinhild Link (Wiesbaden, representing the SSIEM Dieticians Group), Harikleia Ioannou (University A’Pediatric Department, Metabolic Laboratory, ‘Hippocration’ General Hospital of Thessaloniki), Zarifis Dimitroulis (KRIKOS ZOIS – Society for patients and friends of patients with inherited metabolic diseases), Evridiki Drogari (University of Athens, Aghia Sophia Children's Hospital, Unit

• f Metabolic Diseases, Athens), Renza Barbon Galluppi (UNIAMO

FIMR, Italy), Susan Udina (COMETA ASMME – Associazione Studio Malattie Metaboliche Ereditarie – ONLUS, Italy), Hanka Meutgeert (Volwassenen en Kinderen met Stoffwisselingsziekten [VKS], Nether- lands), Vanessa Ferreira (Associação Portuguesa CDG, Portugal), Miguel Macedo (Apofen, Portugal), Sérgio Braz Antão (Rarrisimas, Portugal), Sergi Faber (Catalana de Trastorns Metabòlics Hereditaris, Spain), Sofia Nordin (Svedish Orphan Biovitrium AB [SOBI], Sweden), and Steven Hannigan (CLIMB, Children Living with Inherited Metabolic Diseases, National Information Centre for Metabolic Diseases, and EMDA, the European Metabolic Disorders Alliance). This publication arises from the project “European registry and net- work for intoxication type metabolic diseases” (E-IMD; EAHC no 2010 12 01) which has received funding from the European Union, in the framework of the Health Programme. After the end of the EU funding period the E-IMD patient registry will be sustained by funding from the Kindness-for-Kids Foundation (Munich, Germany).

• M. Baumgartner, J. Häberle and A. Laemmle (Zurich, Switzerland) are

supported by radiz – Rare Disease Initiative Zurich, a clinical research priority programme of the University of Zurich. Drs Murphy and Lachmann were supported by the National Institute for Health Research University College London Hospitals Biomedical Research Centre. Compliance with ethics guidelines Conflict of interest none. Human and animal rights and informed consent All procedures followed were in accordance with the ethical standards of the responsible committee on human studies (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000. Informed consent was

• btained from all patients or their legal guardians prior to being included

in the study in countries where this was needed by law. This article does not contain animal subjects.

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J Inherit Metab Dis

SLIDE 16

• F. Gleich I G. Haege I C. Staufner I P. Burgard

Department of General Pediatrics, Division of Inherited Metabolic Diseases, University Children’s Hospital Heidelberg, Im Neuenheimer Feld 430, D-69120 Heidelberg, Germany

• A. G. Cazorla I E. C. i Saladelafont I C. Ortez

Servicio de Neurologia and CIBERER, ISCIII, Hospital San Joan de Deu, Barcelona, Spain

• V. Valayannopoulos I J.-B. Arnoux I P. de Lonlay

Hôpital Necker-Enfants Malades, Assistance Publique-Hôpitaux de Paris, Reference Center for Inherited Metabolic Disease, Necker-Enfants Malades University Hospital and IMAGINE Institute, Paris, France

• A. M. Lund I L. Aksglaede

Centre for Inherited Metabolic Diseases, Department of Clinical Genetics, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark

• A. B. Burlina I F. Furlan

U.O.C. Malattie Metaboliche Ereditarie, Azienda Ospedaliera di Padova, Padova, Italy

• J. Sykut-Cegielska

Screening Department, Institute of Mother and Child, Warsaw, Poland

• F. A. Wijburg I E. Langereis

Department of Pediatrics, Academisch Medisch Centrum, Amsterdam, Netherlands

• E. L. Teles I E. Rodrigues

Unidade de Doenças Metabólicas, Serviço de Pediatria, Hospital de

• S. João, EPE, Porto, Portugal
• J. Zeman I V. Dvorakova

First Faculty ofMedicine, Charles University andGeneral University

• f Prague, Prague, Czech Republic
• C. Dionisi-Vici I D. Martinelli

U.O.C. Patologia Metabolica, Ospedale Pediatrico Bambino Gésu, Rome, Italy

• I. Barić

School of Medicine, University Hospital Center Zagreb and University of Zagreb, Zagreb, Croatia

• D. Karall I S. Scholl-Bürgi

Clinic for Pediatrics I, Inherited Metabolic Disorders, Medical University of Innsbruck, Innsbruck, Austria

• P. Augoustides-Savvopoulou

1st Pediatric Department, Metabolic Laboratory, General Hospital of Thessaloniki ‘Hippocration’, Thessaloniki, Greece

• P. Avram

Institute of Mother and Child Care “Alfred Rusescu”, Bucharest, Romania

• M. R. Baumgartner I J. Häberle I A. Laemmle

Division of Metabolism and Children’s Research Centre, University Children’s Hospital Zurich, Steinwiesstraße 75, CH-8032 Zurich, Switzerland

• J. Blasco-Alonso

Hospital Materno-Infantil (HRU Carlos Haya), Málaga, Spain

• B. Chabrol

Centre de Référence des Maladies Héréditaires du Métabolisme, Service de Neurologie, Hôpital d’Enfants, CHU Timone, Marseilles, France

• A. Chakrapani

Birmingham Children’s Hospital NHS Foundation Trust, Steelhouse Lane, Birmingham B4 6NH, UK

• K. Chapman I M. L. Summar

Children’s National Medical Center, 111 Michigan Avenue, N.W., Washington, DC 20010, USA

• M. L. Couce

Metabolic Unit, Department of Pediatrics, Hospital Clinico Universitario de Santiago de Compostela, Santiago de Compostela, Spain

• L. de Meirleir

University Hospital Vrije Universiteit Brussel, Bruxelles, Belgium

• D. Dobbelaere

Centre de Référence des Maladies Héréditaires du Métabolisme de l’Enfant et de l’Adulte, Hôpital Jeanne de Flandre, Lille, France

• W. Gradowska

Department of Laboratory Diagnostics, The Children’s Memorial Health Institute, Warsaw, Poland

• S. Grünewald I N. Thompson

Metabolic Unit Great Ormond Street Hospital and Institute for Child Health, University College London, London, UK

• A. Jalan

N.I.R.M.A.N., Om Rachna Society, Vashi, Navi Mumbai, Mumbai, India

• R. Lachmann

Charles Dent Metabolic Unit, National Hospital for Neurology and Neurosurgery, London, UK

• S. Matsumoto

Department of Pediatrics, Kumamoto University Hospital, Kumamoto City, Japan

• C. Mühlhausen

Klinik für Kinder- und Jugendmedizin, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany

• H. O. de Baulny

Hôpital Robert Debré, Université de Paris, Paris, France

• D. P. Ramadža

University Hospital Center Zagreb, Zagreb, Croatia J Inherit Metab Dis

SLIDE 17

• L. Peña-Quintana

Hospital UniversitarioMaterno-Infantil de Canarias, Unit of Pediatric Gastroenterology, Hepatology and Nutrition, University of Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, Spain

• E. Sokal

Service Gastroentérologie and Hépatologie Pédiatrique, Cliniques Universitaires St Luc, Université Catholique de Louvain, Bruxelles, Belgium

• R. Vara

Evelina Children’s Hospital, St Thomas’ Hospital, London, UK

• I. V. Pinera

Inborn Metabolic Disease Unit, Hospital Virgen de la Arrixaca de Murcia, El Palmar, Spain

• J. H. Walter

Manchester Academic Health Science Centre, Willink Biochemical Genetics Unit, Genetic Medicine, University of Manchester, Manchester, UK

• M. Williams

Erasmus MC-Sophia Kinderziekenhuis, Erasmus Universiteit Rotterdam, Rotterdam, Netherlands J Inherit Metab Dis