Study of Inborn Errors of Metabolism in Urine from Patients with Unexplained Mental Retardation

J Inherit Metab Dis (2010) 33:1–7 DOI 10.1007/s10545-009-9004-y

ORIGINAL ARTICLE

Study of inborn errors of metabolism in urine from patients with unexplained mental retardation

Angela Sempere & Angela Arias & Guillermo Farré & Judith García-Villoria & Pilar Rodríguez-Pombo & Lurdes R. Desviat & Begoña Merinero & Angels García-Cazorla & Maria A. Vilaseca & Antonia Ribes & Rafael Artuch & Jaume Campistol

Received: 6 August 2009 /Revised: 8 October 2009 /Accepted: 2 November 2009 /Published online: 5 January 2010 # SSIEM and Springer 2009

Abstract Mental retardation (MR) is a common disorder such as determination of creatine (Cr) metabolites, acylcar- frequently of unknown origin. Because there are few studies nitines, purine, and pyrimidines in urine were applied. We regarding MR and inborn errors of metabolism (IEM), we found seven patients with IEM [three with cerebral Cr aimed to identify patients with IEM from a cohort of 944 deficiency syndromes (CCDS)], one with adenylosuccinate patients with unexplained MR. Biochemical examinations lyase (ADSL) deficiency, and three, born before the neonatal metabolic screening program in Catalonia, with phenylketonuria (PKU). All told, they represent 0.8% of the whole cohort. All of them had additional symptoms such as Communicated by: Georg Hoffmann epilepsy, movement disorders, autism, and other psychiatric References to electronic databases: OMIM 602360, OMIM 601240, disturbances. In conclusion, in patients with MR, it is essential OMIM 300352, OMIM 103050, OMIM 274270, OMIM 300322, OMIM to perform a thorough appraisal of the associated signs and 258900, OMIM 261000, OMIM 311250, OMIM 606054, OMIM 60983, symptoms, and in most disorders, it is necessary to apply OMIM 611935, OMIM 603921, OMIM 243500, OMIM 231670, OMIM 210200, OMIM 300256, http://www.hgvs.org.mutnomen. specific analyses. In some cases, it is important to achieve an early diagnosis and therapy, which may reduce the morbi- Competing interest: None declared mortality, and to offer genetic counselling. A. Sempere : A. García-Cazorla : J. Campistol Pediatric Neurology, Hospital Sant Joan de Déu, Abbreviations Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), ISCIII, ADSL Adenylosuccinate lyase Esplugues, Spain AGAT Arginine-glycine amidinotransferase : : : CCDS Cerebral creatine deficiency syndromes A. Arias G. Farré J. García-Villoria A. Ribes CDG Congenital disorders of glycosylation Division of Inborn Errors of Metabolism (IBC), Department of Biochemistry and Molecular Genetics, Cr Creatine Hospital Clínic and CIBERER, ISCIII, Crn Creatinine Barcelona, Spain CRTR Creatine transporter deficiency DPD Dihydropyrimidine dehydrogenase P. Rodríguez-Pombo : L. R. Desviat : B. Merinero Centro de Diagnóstico de Enfermedades Moleculares, GAA Guanidinoacetate Departamento de Biología Molecular CBM-SO, GAMT Guanidinoacetate methyltransferase Facultad de Ciencias, Universidad Autónoma and CIBERER, GC/MS Gas chromatography/mass spectrometry ISCIII, ¹H-MRS Proton magnetic resonance spectroscopy 28049 Madrid, Spain HPLC High-pressure liquid chromatography M. A. Vilaseca : R. Artuch (*) HPLC-MS/MS Electrospray ionization tandem mass Departamento de Bioquímica Clínica, spectrometry Hospital Sant Joan de Déu, IEM Inborn error of metabolism Passeig Sant Joan de Dèu, 2, 08950 Esplugues, Barcelona, Spain MMACHC Methylmalonic aciduria CbLC e-mail: [email protected] complementation group 2 J Inherit Metab Dis (2010) 33:1–7

MMADHC Methylmalonic aciduria CbLD metabolites, acylcarnitines, and purine and pyrimidines in complementation group urine, were carried out. MR Mental retardation MRI Magnetic resonance imaging OTC Ornithine transcarbamylase Patients and methods PKU Phenylketonuria S-Ado Succinyladenosine A group of 944 unrelated patients with MR of unknown SAICAr Succinylamino-imidazole carboxamide cause living in institutions for mentally handicapped riboside persons was screened. Initially, we selected 38 centers SUCLA-2 Succinyl-coenzyme A (CoA) ligase for mentally handicapped patients; seven refused to UMP Uridine monophosphate participate. Written informed consent was obtained from all families participating in the study. Biochemical and genetic investigations were performed following diagnostic protocols approved by the Ethics Committee of Hospital Sant Introduction Joan de Déu (Barcelona). Patients were classified into two groups: (1) 317 patients with isolated MR; mean age 35 − Mental retardation (MR) affects up to 3% of the population [standard deviation (SD) 17.3, range 5 84) years, 200 males (Curry et al. 1997). Despite its prevalence, our knowledge (63.1%) and 117 females (36.9%); and (2) 627 patients about MR etiology is limited. The spectrum of possible presenting with MR associated with other symptoms, such as causes of this disorder as well as the range of diagnostic epilepsy, movement disorders, sensory deficits, autism, or investigations is very wide. Accurate diagnosis is important other psychiatric disturbances (mean age 33 years, SD 15.6, – because it has implications regarding treatment (in poten- range 3 76). This group was designated MR-plus. The tially treatable disorders), prognosis, genetic counselling of characteristics of this latter group are summarized in Table 1. families, and implementation of prevention programs (Battaglia and Carey 2003). Patients with MR should have Clinical studies a rational evaluation in which the key elements include medical, family, and developmental history; dysmorpho- Clinical data were compiled with diagnosis surveys logic and neurologic examination; and appropriate use of completed by MD and nursing staff, depending on the ′ laboratory and neuroimaging exams (Moeschler 2008). center. The questions were: patient s name, age, gender, However, despite numerous diagnostic efforts, in about degree of mental retardation, language involvement, senso- 50% of cases, the cause of MR remains unexplained ry deficits, epilepsy, movement disorders, autism spectrum (Battaglia and Carey 2003), although some guidelines for disorder, other neuropsychiatric disorders, and patient “ ” metabolic investigations have previously been reported treatments. The item neuropsychiatric disorders included (Curry et al. 1997; García-Cazorla et al. 2009). psychiatric disorders that fulfil clinical criteria of the Inborn errors of metabolism (IEM) remain responsible Diagnostic and Statistical Manual of Mental Disorders, for only about 1% of MR, which is a very low proportion Fourth Edition (DSM IV), such as anxiety, personality, even compared with other causes, such as cytogenetic and mood, psychotic, and impulsive-control disorders. subtelomeric aberrations and fragile X disease (9.5%, 4.4%, Metabolic investigations and 5.4%, respectively) (Van Karnebeek et al. 2005a). Furthermore, few IEMs cause MR as an isolated clinical Random urine samples were collected and stored at −20 C manifestation. The possibility of an underlying metabolic until the moment of the analysis. We performed the disorder increases if MR is associated with other neurological following biochemical analyses: signs, such as psychiatric disturbances, cerebellar dysfunc- tion, and epilepsy (García-Cazorla et al. 2006, 2009). – Urine creatinine: assayed with the Jaffé Kinetic procedure Although IEMs are rare causes of MR, the possibility of (A11A00060, ANX Diagnostic, Montpellier, France). genetic counselling and, more importantly, the application of – Screening of cerebral creatine deficiency syndromes specific treatments in some disorders make the implementa- (CCDS): determination of urine creatine (Cr) and tion of diagnostic protocols necessary. guanidinoacetate (GAA) by high-performance liquid Because there are few studies regarding MR and IEM, chromatography (HPLC) with fluorescence detection we aimed to identify patients with metabolic disorders from (Perkin Elmer, Norwalk CT, USA) and by electrospray a cohort of 944 patients with unexplained mental retarda- ionization tandem mass spectrometry (HPLC-MS/MS) tion living in institutions for mentally handicapped persons (Waters-Micromass, Manchester, UK, model Quatro in Catalonia. Biochemical examinations, such as creatine micro ™ API), as previously reported (Arias et al. 2006). J Inherit Metab Dis (2010) 33:1–7 3

Table 1 Clinical features of the 627 patients with mental retardation – Screening of urea cycle defects: orotic acid quantification plus by HPLC with spectrometric detection (Espinós et al. Clinical features Patients 2009). – Screening of mucopolysaccharidosis: determination of Age at evaluation (mean/SD/range) 33/15.7/3–76 glycosaminoglycans by dimethyl-blue automated spec- Children (< 18 years) 16% (103/627) trometric procedure (Andrade et al. 2008). Gender – Quantitative study of acylcarnitines in urine: performed -Males 65% (411/627) by HPLC-MS/MS according to Tortorelli et al. 2005. -Females 34% (100/627) Quantification of acylcarnitine species was achieved Degree of mental retardation using Neolynx software (Micromass. Model Quatro -Mild 11% (70/627) microTM API, Waters). In addition, analysis of urinary -Moderate 15% (96/627) organic acids by gas chromatography mass spectro- -Severe 19% (123/627) metry (GC/MS) of the samples with abnormal acylcar- -Profound 45% (286/627) nitine profile was performed by the usual method of -Not specified 8% (52/627) solvent extraction and trimethylsilyl derivatization Language involvement (Tanaka et al. 1980). -None or mild 50% (317/627) – Specific metabolic defects screened are listed in Table 2. -Moderate 13% (87/627) -Severe 34% (213/627) After the screening, the studies of positive cases were -Not specified 1.6% (9/627) completed as follows: Sensoria deficits – In cases of CCDS: cerebral Cr content was measured by -None 84% (526/627) proton magnetic resonance spectroscopy (¹H-MRS), as -Vision 7.0% (44/627) previously reported (Newmeyer et al. 2005). Both Cr -Hearing 2.7% (17/627) uptake and guanidinoacetate methyltransferase (GAMT) -Not specified 6.4% (40/627) activity in cultured fibroblast were measured in cases of Epilepsy Cr transporter (CRTR) and GAMT deficiencies respec- -None 54% (338/627) tively, according to previously reported procedures -Controlled 40% (250/627) (Salomons et al. 2001; Stöckler et al. 1996). Genetic -Refractory 5.9% (37/627) analysis of SLC6A8 gene in CRTR deficiency and -Not specified 0.3% (2/627) GAMT gene in GAMT deficiency was performed using Movement disorder fibroblast cell lines as a source of messenger RNA -No 59% (368/627) (mRNA) and genomic DNA (Arias et al. 2007; Sempere -Yes 41% (259/627) et al. 2009)(Table3). Autism spectrum disorder – For the other IEMs detected, such as PKU and -No 71% (448/627) adenylosuccinate lyase (ADSL) deficiency, the next -Yes 28% (161/627) step was the mutational study of the genes involved in -Not specified 2.9 (18/627) the disease (Table 3). Mutational analysis was per- Other neuropsychiatric disorders formed by direct sequencing of the exon and intron -No 59% (373/627) boundaries from mRNA or genomic DNA obtained -Yes 33% (207/627) from fibroblast cell lines or blood samples. Mutation -Not specified 7.5% (47/627) nomenclature was in accordance with the Human Variation Society (http://www.hgvs.org.mutnomen). – Screening of purine and pyrimidine disorders: determination of succinylamino-imidazole carboxamide riboside (SAICAr), succinyladenosine (S-Ado), orotic acid, Results urate, thymine, and uracil in urine by HPLC with spectrometric detection (Espinós et al. 2009). IEMs were identified in seven patients, all of them from group – Screening of phenylketonuria (PKU): Fölling test; 2 (MR-plus), whereas no alterations were detected in group 1 positive cases were confirmed with phenylalanine patients (isolated MR). Three patients were affected with quantification by ion exchange chromatography with CCDS (two brothers, patients 1 and 2, with a CRTR ninhydrin derivatization and spectrometric detection deficiency, and patient 3 with GAMT deficiency). One patient (Artuch et al. 2004). was affected with a purine disorder due to ADSL deficiency 4 J Inherit Metab Dis (2010) 33:1–7

Table 2 Inborn errors of metabolism screened Brain creatine deficiencies -Glycine amidinotransferase (AGAT) deficiency (OMIM 602360) -Guanidinoacetate methyltransferase (GAMT) deficiency (OMIM 601240) -Creatine transporter deficiency (CRTR) (OMIM 300352) Purine and pyrimidine disorders -Adenylosuccinate lyase (ADSL) deficiency (OMIM 103050) -Dihydropyrimidine dehydrogenase (DPD) deficiency (OMIM 274270) -Lesch-Nyhan syndrome (OMIM 300322) -Uridine monophosphate (UMP) synthetase deficiency (OMIM 258900) Phenylketonuria (PKU) (OMIM 261000) Urea cycle defects -Ornithine transcarbamylase (OTC) deficiency (OMIM 311250) Mucopolysaccharidosis Organic acidurias -Propionic acidemia (OMIM 606054). -Methylmalonic acidurias: Mut(0), CblA, CblB, CblC, CblD (OMIM: 251000; 251100; 251110; 277400; 277410). -Isovaleric acidemia: MMACHC (OMIM 609831); MMADHC (OMIM 611935); SUCLA-2 (OMIM 603921). -Isovaleric academia (OMIM 243500). -Glutaric acidemia type I (OMIM 231670). -3-methylcrotonyl-coenzyme A (CoA) carboxylase deficiency (OMIM 210200). -2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency (OMIM 300256). Mitochondrial-β oxidation deficiencies

and three patients with PKU (patients 5 and 6, brothers) ADSL deficiency and three with PKU. We found that IEMs caused by phenylalanine hydroxylase deficiency (patients were present in almost 0.8% of the whole cohort of 944 were older than 25 years of age and were born when newborn mentally retarded patients, which is similar to results screening was not fully implemented in our geographical reported in other studies, varying from 0.2% to 8.4% area). Data from these patients are summarized in Table 3, (median 1%) (Van KarnebeeK et al. 2005b). In these including clinical signs, neuroimaging studies, and diagnosis studies, the higher prevalence was from countries where test results. No mucopolysaccharidosis cases were detected. specific entities are more common, such as aspartylglyco- Urine organic acid analysis disclosed normal results, except saminuria in Finland (Matilainen et al. 1995), or else the for one patient who showed a high isolated excretion of figures were obtained by applying checklist criteria (Hunter methylmalonic acid in two independent urine samples; the 2000; Papavasiliou et al. 2000) reaching an increase of etiology is now under investigation. yield to 13.6% (Papavasiliou et al. 2000). In our study, this It is noteworthy that CCDS diagnosis was initially percentage increased to 1.1% (0.8% considering only index suspectedin39furtherpatientsbasedonalteredbiochemical cases) when it was referred to the group with MR-plus. This findings. In 37 of the 39 cases, this syndrome was finally ruled means that most patients with metabolic disorders have out on the basis of further biochemical, neuroimaging, other symptoms, as has been previously reported (Curry et enzymatic, or molecular studies. One patient, who presented al. 1997; García-Cazorla et al. 2006, 2009; Moog et al. a clearly increased GAA excretion in urine (suspicion of 2008). In this way, all of our patients with metabolic GAMT deficiency), refused further collaboration and a final disorders also had additional disorders, such as epilepsy, diagnosis was not possible, whereas the other patient who movement disorders, autism and other psychiatric distur- presented a decreased GAA value in urine (suspicion of bances, anomalies of cranial perimeter, facial dysmorphia, AGAT deficiency) suddenly died due to cardiorespiratory and brain neuroimaging abnormalities. Considering that arrest. IEMs were also diagnosed in patients with isolated MR, our results would probably not support the notion that only patients with MR-plus are candidates for IEM screening. Discussion Although comparison between studies is not possible given the lack of uniformity of metabolic testing from study We found seven patients with IEM, all in the group of 627 to study (Van Karnebeek et al. 2005a), other studies in patients with MR-plus: three patients with CCDS, one with institutions for mentally retarded persons found more nei ea i 21)33:1 (2010) Dis Metab Inherit J Table 3 Clinical, biochemical, and molecular data of patients identified with inborn errors of metabolism (IEM)

Patient Age Degree Epilepsy Movement Sensory Psychiatric Brain MRI Other signs Biochemical IEM molecular data years MR disorders deficits disorders data urine

1a 27 Profound Controlled with No No Autistic-like Normal Low Cr peak Severe speech disorder Flat ↑ Cr/Crn= CRTR deficiency: mutation antiepileptic drugs behavior in ¹H-MRS malar region 3.0 SLC6A8 gene, c.1079_1081delTCT (p.Phe360del) 2a 40 Profound Controlled with No No Autistic-like Normal Low Cr peak Severe speech disorder ↑ Cr/Crn= CRTR deficiency: mutation

antiepileptic drugs behavior in ¹H-MRS Microcephaly 2.6 SLC6A8 gene; – 7 c.1079_1081delTCT (p.Phe360del) 3 45 Profound Refractory to No No Autistic=like Normal low Cr peak in Severe speech disorder Flat ↑ GAA= GAMT deficiency: antiepileptic drugs behavior, ¹H-MRS malar region Occipital plano 374 mmol/ mutations GAMT gene; hyperactivity mol Crn c.316C>T (p.Gln106stop) c.407C>T (p.Thr136Met) 4 7 Profound Initially no. After Spastic Deafness Autistic-like Brain atrophy Severe speech disorder ↑ S-Ado and ADSL deficiency: diagnosis developed a tetraparesis behavior, Cerebellar atrophy Macrocephaly Facial SAICAr homozygous mutation refractory epilepsy. hyperactiviy White matter dysmorphism Strabismus (ratio=1.88) ADSL gene; c.1107A>C abnormalities Coma vigil (p.I369L) 5a 28 Mild No No No Autistic-like Brain atrophy Mild speech disorder Phe=872 Mutations PAH gene: IVS10 behavior, Occipital white Microcephaly µmol/g IVS2+5G>C hyperactiviy matter abnormalities Crn 6a 36 Moderate No No No Hyperactiviy Brain atrophy Moderate speech disorder Phe=1,358 Mutations PAH gene: IVS10 Occipital white Microcephaly µmol/g IVS2+5G>C matter abnormalities Crn 7 32 Severe No Spastic No Psychosis Brain atrophy Moderate speech disorder Phe=1,780 Mutations PAH gene: tetraparesis Microcephaly µmol/g R243Q L48S Crn a Patients 1 and 2, and patients 5 and 6, were sibs 5 6 J Inherit Metab Dis (2010) 33:1–7 frequent PKUs, urea cycle disorders, and mucopolysacchar- for it is especially important in older patients in whom this idosis (Van Buggenhout et al. 2001; Ryan and Sunada screening was not performed. In our cohort with MR-plus, 1997; Zhang et al. 2000). A recent study suggested a high we had 215 patients older than 25 years, which means that prevalence of congenital glycosilation defects (CDG) in they were born before neonatal metabolic screening started children with MR plus other neurological signs (Van systematically in Catalonia. We found three patients with KarnebeeK et al. 2005b). PKU, all of them older than 25, which results in a As a first step, we used urine samples because of the frequency of 0.47% of all MR-plus patients, and this large population and the ease of obtaining them. The increases to 1.39% if we consider only patients older than limitation of this approach is, however, that it did not allow 25. Interestingly, with PKU being one of the most frequent screening of all urea cycle disorders (except for OTC IEMs, its prevalence among our patients was similar to that deficiency) or CDGs (which are initially diagnosed in of CCDS, although the implementation of the neonatal serum samples), which can present with nonspecific MR screening program surely has led to a reduction of PKU (García-Cazorla et al. 2006, 2009). Moreover, the lack of a prevalence in younger patients. mucopolysaccharidosis diagnosis might also be explained In conclusion, in patients with MR, rather than applying by the fact that we did not apply electrophoretic procedures a standardized metabolic screening, it is essential to for screening for the disorder. perform a thorough appraisal of the signs and symptoms CCDS are a recently described group of IEM that affect associated with MR (epilepsy, autistic traits, psychiatric the synthesis (AGAT and GAMT deficiency) and transport disorders, movement disorders, dysmorphic traits), as in of Cr (CRTR deficiency). The clinical hallmarks of these most disorders, it is necessary to apply specific analyses. In disorders are MR, epilepsy, autistic behavior, and language some cases, it is particularly important to suspect these disorders (Stockler et al. 2007). Some studies have reported diseases in order to achieve an early diagnosis and initiate a high frequency of these disorders in patients with MR therapy, which may reduce the morbimortality, and to offer (Arias et al. 2007; Clark et al. 2006; Lion-Francois et al. genetic counselling in cases when it is indicated. CCDS 2006; Newmeyer et al. 2005). In our study, we found one may be a treatable disorder with an increasing frequency patient with GAMT deficiency and two with CRTR that should be considered. deficiency, which means that CCDS might have a frequency of 0.47% in our population of MR-plus patients, and it Acknowledgements We thank the patients, their families, and the was as frequent as PKU. Synthesis defects can be treated institutions for mentally handicapped persons for their collaboration. with Cr supplements, as in our patient with GAMT The study was financially supported by grants FIS 05/1200, FIS deficiency, resulting in clinical improvement, especially in PI051180, CAIXA GIRONA, and the Alicia Koplowitz Foundation. The groups are funded by the Centro de Investigación Biomédica en improving attention and controlling epilepsy, as has been Red de Enfermedades Raras (CIBERER), ISCIII. previously reported (Mercimek-Mahmutoglu et al. 2006). Up to now, there have been no successful treatment strategies for CRTR deficiency. Our results suggest that screening for CCDS in patients with MR has a high References diagnostic yield, and the diagnosis of Cr synthesis defects is especially important, as they are treatable conditions. 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