Creatine depletion in a new case with AGAT deficiency: clinical and genetic study in a large pedigree

Molecular Genetics and Metabolism 77 (2002) 326–331 www.academicpress.com

Creatine depletion in a new case with AGAT deficiency: clinical and genetic study in a large pedigree Roberta Battini,a Vincenzo Leuzzi,b Carla Carducci,c Michela Tosetti,d Maria C. Bianchi,e €ckler-Ipsiroglu,f and Giovanni Cionia,* Chike B. Item,f Sylvia Sto a

Division of Child Neurology and Psychiatry, IRCCS Stella Maris and University of Pisa, Via dei Giacinti 2, Calambrone Pisa 56018, Italy b Department of Child Neurology and Psychiatry, University ‘‘La Sapienza’’, Rome, Italy c Department of Experimental Medicine and Pathology, University ‘‘La Sapienza’’, Rome, Italy d IRCCS Stella Maris, Pisa, Italy e Department of Neuroradiology, Santa Chiara Hospital, Pisa, Italy f Department of Pediatrics, University Hospital and General Hospital of Vienna, Austria Received 22 August 2002; received in revised form 26 September 2002; accepted 26 September 2002

Abstract Arginine:glycine amidinotransferase (AGAT, EC 2.1.4.1) deficiency is a recently recognized autosomal recessive inborn error of creatine biosynthesis, characterized by mental retardation and severe language impairment. We extensively investigated a third 5year-old patient with AGAT deficiency, discovered in the pedigree of the same Italian family as the two index cases. At the age of 2 years he presented with psychomotor and language delay, and autistic-like behavior. Brain MRI was normal, but brain 1 H-MRS disclosed brain creatine depletion, which almost completely normalized following creatine monohydrate supplementation. A remarkable clinical improvement paralleled the restoration of brain creatine concentration. AGAT and GAMT (guanidinoacetate:methyltransferase) genes were analyzed in the proband and in 26 relatives, including the two cousins with AGAT deficiency. Sequencing of the probandÕs AGAT gene disclosed the same homozygous mutation at nt position 9093 converting a tryptophan (TGG) to a stop codon (TAG) at residue 149 (W149X), as already described in the two previously reported cases. The probandÕs parents and 10 additional subjects of the pedigree were carriers for this mutation. AGAT deficiency was further confirmed by undetectable AGAT activity in the patientÕs lymphoblasts. Mutation analysis of the GAMT gene revealed a sequence variation in exon 6 (T209M), not in the proband, but in 15 additional subjects from the pedigree. The silent nature of this sequence variation is supported by its homozygosity in one AGAT deficient cousin and in one asymptomatic adult, both with normal GAMT activity. Ó 2002 Published by Elsevier Science (USA). Keywords: Guanidinoacetate methyltransferase (GAMT); Arginine:glycine amidinotransferase (AGAT); Creatine deficiency; Genetical analysis; Proton spectroscopy

Introduction Creatine (Cr) and creatine phosphate (CrP) play essential roles in the storage and transmission of phosphate-bound energy. Creatine is synthesized mainly in the liver and pancreas by a two-step reaction. The first is catalyzed by arginine:glycine amidinotransferase (AGAT, EC 2.1.4.1), the limiting enzyme whose expression is repressed by creatine, the second by guani-

*

Corresponding author. Fax: +39-050-32214. E-mail address: [email protected] (G. Cioni).

dinoacetate-methyltransferase (GAMT, EC 2.1.1.2). Cr is not utilized in these organs but concentrated in tissues with a high activity of creatine kinase (CK) (such as muscle and brain) by means of an active Naþ and an energy-dependent creatine transporter system (CT1). CK catalyzes the phosphorylation and dephosphorylation of Cr and CrP and thus provides a high-energy phosphate buffering system during states of ATP synthesis and ATP utilization [1,2]. Despite the importance of this metabolic pathway no primary metabolic disorders of Cr synthesis were recognized until 1994, when the first case of GAMT deficiency was described [3]. Since then a few more sporadic

1096-7192/02/$ - see front matter Ó 2002 Published by Elsevier Science (USA). PII: S 1 0 9 6 - 7 1 9 2 ( 0 2 ) 0 0 1 7 5 - 0

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cases have been reported [4–8]. A comprehensive review of this disease has recently been published [9]. A second, X-linked, inherited alteration of Cr metabolism, caused by the derangement of CT1 in the central nervous system level has been reported in two pedigrees [10,11]. Finally, two Italian sisters with a previously unclassified deficit of brain creatine [12] turned out to be affected by AGAT deficiency [13]. We extensively investigated a third patient with AGAT deficiency, discovered in the family of the two index cases [12,13]. In this work we report his clinical, biochemical, and molecular results, as well as mutation studies of the AGAT and GAMT genes in the whole family pedigree.

Patient and pedigree study Fig. 1 shows the family pedigree, whose origins are in the South of Italy (Calabria). The propositus (IV:7), now 5 years old, is a second cousin of the two previously described sisters with AGAT deficiency (IV:4, IV:5) [12,13] and he is the first son of healthy Italian parents who report no known consaguinity. Subjects III:23 (probandÕs father) and III:9 (father of index patients 1 and 2) are first cousins. The child was admitted to the Division of Child Neurology and Psychiatry of Pisa at the age of 2 with psychomotor delay, absence of language, and behavioral disorders. Body height and weight were normal, but head circumference was mildly reduced (47 cm, )1 SD). He showed a severe developmental retardation (Griffith Developmental Scales (GDS): developmental quotient (DQ) <50; Uzgiris–Hunt Test (U–H): fourth sensorimotor stage), more pronounced in language and communication domains. Additional findings included a mild generalized hypotonia, poor social contact, short attention span, and stereotypic movements of the hands. Asleep and awake EEGs were normal. Chromosomal evaluation revealed a normal male karyotype and

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fragile-X disease was also ruled out. Normal neurometabolic analyses included: plasma and urine amino acids, serum lactate and pyruvate, urine organic acids, and oligosaccharides and mucopolysaccharides. Since brain Cr depletion had previously been detected in his two cousins, Cr metabolism was extensively investigated. Brain magnetic resonance imaging (MRI) and proton magnetic resonance spectroscopy (1 H-MRS) were performed with a 1.5 T clinical MR scanner (GE Signa Horizon 1.5, Milwaukee, WI), using the same methodology reported in the index cases [12]. Images were normal but 1 H-MRS revealed a total absence of Cr/CrP peak in the paraventricular white matter, in the parieto-occipital cortex and in the cerebellum (Fig. 2a). When biochemical and genetic investigations confirmed AGAT deficiency, the child was put on oral creatine monohydrate treatment at a dose of 400 mg/kg/ day. Serial brain 1 H-MRS were performed during a 2year period of therapy, demonstrating a progressive restoration of brain Cr signal (Figs. 2b and c). Clinically, three months after the beginning of treatment, the child showed a clear improvement. He paid more attention to verbal messages, understood simple verbal commands, and exhibited more communicative intent and, successively, at the age of 3 years, he had learned to speak with simple sentences. At the last examination, at the age of 5 (3 years after the beginning of Cr supplementation), the child was mildly retarded (GDS DQ: 63) with a persistent lag in language development, but a marked improvement in interpersonal interaction and a more protracted attention span.

Molecular and biochemical investigations: methods and results Genetic analysis Before the diagnosis of AGAT deficiency was made in the index subjects [12,13], the possibility of a GAMT

Fig. 1. Family pedigree.

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Fig. 2. Time course of brain Cr restoration in a patient with AGAT deficiency. Representative spectra of parietal occipital cortex before (a) and after 12 months (b) of Cr-intake demonstrating an almost complete normalization of brain Cr-signal amplitude (Cr at 3.05 ppm) [single voxel short echo time (TE) stimulated echo acquisition mode technique (STEAM)]. The trend was monitored by 1 H-MRS in a 2-year period of Cr oral intake at doses of 400 mg/kg/day (c). The slight deflection of brain Cr content during the second year of treatment was probably due to inadequate therapy compliance.

gene involvement had been also considered. Therefore, both GAMT and AGAT loci were analyzed in 27 members of the pedigree. The 6 exons and intron/exon boundaries of the GAMT gene were analyzed by using direct sequencing, as previously described [14]. The 9 exons of the AGAT gene were studied by using PCR followed by direct sequencing. Amplification reactions were performed for 35 cycles with denaturation at 95 °C for 1 min and extension at 72 °C for 1 min. Annealing conditions, MgCl2 concentrations, and primer sequences are shown in Table 1. Sequencing reactions were carried out by using Big Dye Terminator Cycle Sequencing (Applied Biosystem) and electrophoresed on

an ABI PRISM 310 automated sequencer (PE Biosystem). GAMT gene analysis disclosed a single nucleotide alteration in exon 6 at nt position 4024C > T, changing the triplet ACG at position 209 into ATG resulting in an amino acid change from threonine to methionine. The T209M sequence variation was carried by 13 subjects in heterozygous and by 2 subjects in homozygous form (Table 2). No sequence variation in the GAMT gene was found in the proband (IV:7). However, the two cousins (IV:4 and IV:5) previously reported with AGAT deficiency [13], carried the GAMT sequence variation in homozy-

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Table 1 Primer sequence and length, annealing temperature, and Mgþþ concentrations used in PCR for the analysis of AGAT gene Primers

Length (bp)

Annealing conditions

Mgþþ (mM)

Exon 1 for: 50 -ACCTGTTCCCGGCAGCCAAT-30 Exon 1 rev: 50 -CCGCAGGATCGAGTGAGTCA-30

300

60 °C  3000

3.5

Exon 2 for: 50 -TACTCCATCTCCACTTCCTC-30 Exon 2 rev: 50 -AAGCAGTCAGAGGGTAGCAG-30

370

55 °C  1 min

2.25

Exon 3 for: 50 - ACTGCCCTATGAACCTATCATG-30 Exon 3 rev: 50 -CTGGCAGTTTAGTTATGTGAC-30

296

55 °C  1 min

2.25

Exon 4 for: 50 -ACACTACTGAAGTTGTCATGG-30 Exon 4 rev: 50 -AGGTATCAGAGAATCTCTATC-30

347

53 °C  1 min

2.25

Exon 5 for: 50 -CCAGTGCATTGTGTGTGTTCA-30 Exon 5 rev: 50 -CCTAGGTAGTTTGTAACCTGA-30

351

53 °C  1 min

2.25

Exon 6 for: 50 -GCAGGCTGCCTCTGAAATTT-30 Exon 6 rev: 50 -GCAGGCTGCCTCTGAAATTT-30

200

55 °C  1 min

2.25

Exon 7 for: 50 -CTG ATAAGGGCTATTAGAGAC-30 Exon 7 rev: 50 -TAGGAGAAAGATCCTTGGTCA-30

211

55 °C  1 min

2.25

Exon 8 for: 50 -CACATGGAAAGTGAACTACTG-3 Exon 8 rev: 50 -TAACTTGGTGCCTCTAAAAGG-30

367

53 °C  1 min

2.25

Exon 9-S1 for: 50 -GAGAATCGTCATGTTAGTCCA-30 Exon 9-S1 rev: 50 -AAACCAGGCTTACGACCCCT-30

305

55 °C  1 min

2.25

Exon 9-S2 for: 50 -AGTGCATGAACTGTAGTGCTT-30 Exon 9-S2 rev: 50 -ACATTTCAAGCT GCCTTTTGG-30

280

55 °C  1 min

2.25

Table 2 Results of mutation analysis in GAMT and AGAT genes Patient

GAMT gene

AGAT gene

II:1 II:2 II:3 II:4 II:6 II:7 III:2 III:4 III:5 III:6 III:7 III : 8 III : 9 III:10 III:12 III:16 III:18 III:19 III:21 III : 22 III : 23 III:24 III:26 III:27 IV : 4 IV : 5 IV : 7

T209M/WT T209M/WT T209M/WT T209M/WT WT/WT WT/WT WT/WT T209M/WT T209M/WT WT/WT WT/WT T209M=WT T209M=T209M WT/WT T209M/WT WT/WT WT/WT WT/WT WT/WT WT=WT T209M=WT T209M/WT T209M/WT T209M/WT T209M=T209M T209M=WT WT=WT

WT/WT W149X/WT WT/WT W149X/WT WT/WT W149X/WT W149X/WT WT/WT W149X/WT WT/WT W149X/WT W149X=WT W149X=WT W149X/WT WT/WT WT/WT WT/WT WT/WT WT/WT W149X=WT W149X=WT WT/WT W149X/WT WT/WT W149X=W149X W149X=W149X W149X=W149X

AGAT patients and their parents are underlined. See pedigree for identification of analyzed subjects. (WT, wild-type).

gous (IV:4) and heterozygous (IV:5) form, respectively. Furthermore, their father (III:9), who is clinically asymptomatic and had normal neuropsychological development in his childhood, was homozygous for the same mutation. The normality of GAMT activity confirmed that this was a silent variation without obvious consequences on the enzyme protein function (see the following sections) [13]. AGAT gene analysis showed the mutation at nt position (9093A > G) converting a tryptophan (TGG) to a stop codon (TAG) at residue 149 (W149X). The mutation was homozygous in the proband (IV:7) as well as in his two previously reported cousins [13] (IV:4, IV:5). His parents and 10 additional subjects of the pedigree were found to be carriers for W149X mutation (Table 2). The mutation lies within the third exon and theoretically would result in a 274 residue shorter product, but a mRNA nonsense mediated decay process may be hypothesized [13]. Biochemical investigation Guanidinoacetate (GAA) is the product of AGAT and substrate for GAMT. Quantitative determination of urinary GAA concentrations [15], which are high in patients with GAMT deficiency [8,16], revealed a very low level (36 lmol/L, n.v. 60–850), as in the previous index cases [12]. Creatinine (Crn) level in a urine

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Table 3 AGAT and GAMT activities (given in nmol/h/mg protein) and respective gene mutations in the proband, his parents, his previously reported cousins with AGAT deficiency and their parents

Patient (IV:7), proband Father (III:6), proband Mother (III:7), proband Patient IV:4½11 , index patient 1 Patient IV:5½11 , index patient 2 Father III:9½11 , index patients 1 and 2 Mother III:8½11 , index patients 1 and 2 Controls

AGAT activity and mutation

GAMT activity and mutation

<0.3, W149X/W149X n.d., W149X/WT n.d., W149X/WT <0.3, W149X/W149X <0.3, W149X/W149X 4.9, W149X/WT 6.3, W149X/WT 12.6–23.4 WT/WT

0.62, WT/WT n.d., WT/WT n.d., T209M/WT 0.53, T209M/T209M 0.64, T209M/WT n.d., T209M/T209M n.d., T209M/WT 0.61–0.84 WT/WT

n.d., not determined.

sample was within the normal range (3350 mol/L, n.v. 1800–4400) [16]. AGAT and GAMT enzyme activities were measured in virus-transformed lymphoblasts, as already described [13,17] (Table 3). AGAT activity was clearly deficient in the proband who is homozygous for the W149X AGAT mutation. As indicated above, he was also homozygous for the wild-type sequence of the GAMT gene and his GAMT activity was normal. GAMT activity was also normal in the two previously described AGAT index patients, who, in addition to the homozygous W149X AGAT mutation, were respectively, homozygous and heterozygous for the T209M sequence variation in the GAMT gene. Despite homozygosity for T209M, GAMT activity was also normal in subject III:9 [13].

Discussion We have reported the third case of AGAT deficiency in a patient belonging to the same pedigree as the only two other cases so far reported [12,13]. As in the two index cases, an AGAT disorder in our proband was clearly demonstrated by undetectable enzyme activity and by the 1 H-MRS result and it was confirmed by molecular genetic testing. Molecular analysis was carried out for AGAT but also for GAMT gene and it was extended to all pedigree subjects because preliminary results showed a GAMT sequence variation in the family pedigree; in addition, GAMT and AGAT affect Cr synthesis and an additional mutation in GAMT gene could play a role in the expression of the AGAT phenotype. The results were puzzling, since the occurrence of these changes in GAMT and AGAT genes was revealed in a large number of subjects. The pattern of disease segregation, the biochemical abnormalities in affected subjects (lack of GAA accumulation in biological fluids and brain creatine depletion, restored by the creatine supplementation), and enzymatic analysis of GAMT and AGAT, all enabled us to attribute a pathogenetic role to the AGAT mutation, while the GAMT sequence variation (even if resulting in a change from a highly conserved

polar amino acid—Thr—into a hydrophobic amino acid—Met) turned out to be silent. The W149X mutation on AGAT genes has already been reported [13] and shows the characteristics of a severe mutation, since the stop codon is at the beginning of the protein and, in the index patients [13] resulted in an absence of messenger RNA. The clinical presentation of AGAT deficiency is rather nonspecific, being characterized by psychomotor delay during the first years of life, mild to moderate mental retardation and, later, severe language delay [12]. Apart from a unique episode of febrile seizures in one subject [12] neither epilepsy nor movement disorders have been reported. A slow somatic growth (head, weight, and length <2 SD) was noticed in the two index cases, while only a mild microcephaly has been detected in our case. Brain MRI was normal and only 1 H-MRS examinations reveal brain Cr depletion in all cases. Cr monohydrate therapy resulted in rapid neurological progress that paralleled the increase in brain creatine concentration. Clinical improvement concerned visuo-perceptual and fine motor skill functions as well as behavioral disorders, although general cognitive competencies and language development remained severely involved [12]. A normalization of somatic growth has also been observed in the two index cases. During therapy, the brain Cr restoration trend was monitored (as in the index cases) by 1 H-MRS and it was similar to that observed in the affected cousins, reaching 90% of normal values. With the discovery of AGAT deficiency, the panorama of inborn errors of Cr metabolism, as predicted on the basis of present knowledge of Cr metabolism and transport, has been completed. Two diseases, GAMT and AGAT deficiencies, are transmitted as autosomal recessive disorders, whereas one, the CT1 transporter defect, is X-linked. Children with brain creatine deficiency present with symptoms that are quite common in many neurological disorders, such as mental retardation, language disorders, epilepsy, autistic-like behavior, neurological deterioration, and movement disorders, which often remain without a definite etiologic diagnosis. The positive

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results of Cr treatment (in GAMT and AGAT defects) and the observation that fetal and early postnatal development is normal in these patients, both support the hypothesis that an early diagnosis and treatment can substantially improve their final prognosis. The occurrence of early severe epilepsy, developmental delay/arrest, neurological regression and movement disorders reported in most of the subjects with GAMT deficiency suggests a severe neurological disorder. The presence of these symptoms should give rise to a complete neurometabolic and neuroradiological assessment, including brain 1 H-MRS examination and determination of GAA in body fluids. In patients affected by AGAT and CT1 deficits, on the other hand, the clinical presentation is quite vague, hardly suggesting a neurometabolic disorder. From a pathogenetic point of view, mental retardation and severe language disorders (the key-symptoms in AGAT and CT1 defects) seem to be the result of a simple depletion of brain Cr, while movement disorders, epilepsy, and neurological deterioration, which characterize a GAMT defect, all point to a possible neurotoxic effect of GAA or other guanidine compounds (increased only in this disease). In conclusion, the possibility of an AGAT deficiency should be considered in all children affected by unexplained mental retardation and severe language delay. Availability and cost of the 1 H-MRS technique are limiting factors for early diagnosis and appreciation of this new, treatable, inherited metabolic disorder. The assessment of Cr and guanidino compounds in biological fluids may be a good alternative strategy for the diagnostic screening of symptomatic subjects.

Acknowledgments The authors would like to thank M.G. Alessandrı, G. De Vito, and S. Bargagna for laboratory and clinical examinations of the proband, and Paul Morse for reviewing the English of the manuscript. This research was supported by Grants RC 1/01 and RC 4/01 from the Italian Ministry of Health, and by the Austrian National Bank Scientific Trust Grants ONB 8676 (to CBI).

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