3 - Creatine deficiency syndromes

Creatine deficiency syndromes are a newly described group of inborn errors of creatine synthesis (arginine:glycine amidinotransferase (AGAT) deficiency and guanidinoacetate methyltransferase (GAMT) deficiency) and of creatine transport (creatine transporter (CRTR) deficiency). The common clinical feature of creatine deficiency syndromes is mental retardation and epilepsy suggesting main involvement of cerebral gray matter. The typical biochemical abnormality of creatine deficiency syndromes is cerebral creatine deficiency, which is demonstrated by in vivo proton magnetic resonance spectroscopy. Measurement of guanidinoacetate in body fluids may discriminate between the GAMT (high concentration), AGAT (low concentration) and CRTR (normal concentration) deficiencies. Further biochemical characteristics include changes in creatine and creatinine concentrations in body fluids. GAMT and AGAT deficiency are treatable by oral creatine supplementation, while patients with CRTR deficiency do not respond to this type of treatment. The creatine deficiency syndromes are underdiagnosed, so their possibility should be considered in all children affected by unexplained mental retardation, seizures and speech delay.

Currently there are 91 treatable inborn errors of metabolism that cause intellectual developmental disorders. Cerebral creatine deficiencies (CDD) comprise three of these: arginine: glycine amidinotransferase [AGAT], guanidinoacetate methyltransferase [GAMT], and X-linked creatine transporter deficiency [SLC6A8]. Intellectual developmental disorder and cerebral creatine deficiency are the hallmarks of CDD. Additional clinical features include prominent speech delay, autism, epilepsy, extrapyramidal movement disorders, and signal changes in the globus pallidus. Patients with GAMT deficiency exhibit the most severe clinical spectrum. Myopathy is a distinct feature in AGAT deficiency. Guanidinoacetate (GAA) is the immediate product in the creatine biosynthetic pathway. Low GAA concentrations in urine, plasma, and cerebrospinal fluid are characteristic diagnostic markers for AGAT deficiency, while high GAA concentrations are characteristic markers for GAMT deficiency. An elevated ratio of urinary creatine /creatinine excretion serves as a diagnostic marker in males with SLC6A8 deficiency. Treatment strategies include oral supplementation of high-dose creatine-monohydrate for all three CDD. Guanidinoacetate-reducing strategies (high-dose ornithine, arginine-restricted diet) are additionally employed in GAMT deficiency. Supplementation of substrates for intracerebral creatine synthesis (arginine, glycine) has been used additionally to treat SLC6A8 deficiency. Early recognition and treatment improves outcomes. Normal outcomes in neonatally ascertained siblings from index families with AGAT and GAMT deficiency suggest a potential benefit of newborn screening for these disorders.

Background Previous reports have demonstrated electrocardiographic and echocardiographic abnormalities consistent with early–stage cardiomyopathy, in patients with creatine transporter deficiency (CRT–D), but cardiac involvement has never been accurately characterized. Our aim was to widen the current knowledge of the cardiac phenotype associated with creatine deficiency in a murine transgenic model and in patients with creatine deficiency syndrome (CDS). Methods Wild type (n = 6) and transgenic CRT–/y mice (n = 6) were evaluated in vivo by electrocardiogram (EKG) and echocardiography for six months, and ex vivo by histological and biochemical analyses. Nine patients with CDS (4 with L–arginine:glycine amidinotransferase deficiency – AGAT–D, 5 with CRT–D) underwent blood samples, including cardiac biomarkers, EKG, Holter monitoring, echocardiography and cardiac magnetic resonance (CMR). Results Compared to wild–type, CRT–/y mice showed a prolongation of QT interval (p = 0.008) (Fig. 1), as well as a trend of reduction of mitochondrial function (Fig. 2). No differences were dected in the left ventricular systolic function, in terms of ejection fraction and global longitudinal strain, at different times. Aldosterone levels were also not different between the two groups. Three patients with CDS also showed a prolonged QTc interval (median QTc 515 ms), while other 3 had a borderline QTc at Holter monitoring, without tachyarrhythmias. No subject had abnormal systolic and diastolic function. At CMR, an increase of native T1 value was reported in 4 subjects (median 1076 ms), 1 with an increase of extracellular volume matching with the detection of late gadolinium enhancement. Finally, 7 subjects with CDS presented with biohumoral signs of neurohormonal activation (2 with elevated renin, 1 with increased aldosterone and 4 with increased norepinephrine levels). Familiar carriers (3 for AGAT–D and 1 for CRT–D) showed no notable cardiac functional alterations, except for QTc prolongation in 2 AGAT–D carriers. Conclusion Prolonged QT interval is observed in a murine model of CRT–D deficiency and in patients with CDS, in absence of an arrhythmic burden. No alterations in cardiac function were detected in the two setting, except for an increase of native T1 in patients with CDS suggesting an interstitial and replacement fibrosis.

Prospective identification by neonatal screening of patients with guanidinoacetate methyltransferase deficiency

Disorders of creatine metabolism arise from genetic alterations of arginine:glycine amidinotransferase (AGAT), guanidinoacetate methyltransferase (GAMT), and the creatine transporter. We developed a strategy for the detection of AGAT and GAMT defects by measurement of guanidinoacetate (GAA) and creatine plus creatinine (Cr+Crn) in biological fluids. Three patients with AGAT deficiency from the same pedigree and their eight relatives, as well as a patient affected by a GAMT defect and his parents were analyzed by a new HPLC procedure in comparison with 90 controls. The method, which uses precolumn derivatization with benzoin, separation with a reversed-phase column, and fluorescence detection, has shown good precision and sensitivity and requires minimal sample handling. In the three AGAT patients, plasma GAA was 0.01-0.04 micro mol/L [mean (SD) for neurologically normal controls was 1.16 (0.59) micromol/L], Cr+Crn was 15-29 micro mol/L [reference limit in our laboratory, 79 (38) micromol/L]. Urinary GAA was 2.4-5.8 micro mol/L [reference, 311 (191) micromol/L], and Cr+Crn was 2.1-3.3 mmol/L [reference, 9.9 (4.1) mmol/L]. We found a smaller decrease in GAA and Cr+Crn in some carriers of an AGAT defect. In the patient with GAMT deficiency, plasma and urine GAA was increased (18.6 and 1783 micromol/L, respectively), and Cr+Crn was decreased in plasma (10.7 micromol/L) and urine (2.1 mmol/L). GAA was increased in the parents' plasmas and in the mother's urine. The assessment of GAA is a new tool for the detection of both GAMT and AGAT deficiencies.

Guanidinoacetate methyltransferase deficiency (GAMT-D) is one of three cerebral creatine (Cr) deficiency syndromes due to pathogenic variants in the GAMT gene (19p13.3). GAMT-D is characterized by the accumulation of guanidinoacetic acid (GAA) and the depletion of Cr, which result in severe global developmental delay (and intellectual disability), movement disorder, and epilepsy. The GAMT knockout (KO) mouse model presents biochemical alterations in bodily fluids, the brain, and muscles, including increased GAA and decreased Cr and creatinine (Crn) levels, which are similar to those observed in humans. At the behavioral level, only limited and mild alterations have been reported, with a large part of analyzed behaviors being unaffected in GAMT KO as compared with wild-type mice. At the cerebral level, decreased Cr and Crn and increased GAA and other guanidine compound levels have been observed. Nevertheless, the effects of Cr deficiency and GAA accumulation on many neurochemical, morphological, and molecular processes have not yet been explored. In this review, we summarize data regarding behavioral and cerebral GAMT KO phenotypes, and focus on uncharted behavioral alterations that are comparable with the clinical symptoms reported in GAMT-D patients, including intellectual disability, poor speech, and autistic-like behaviors, as well as unexplored Cr-induced cerebral alterations.

The objective of the present study was to investigate the effects of the addition of fibre and the antioxidant N-acetylcysteine (NAC) to fat-rich diets on fetal intrauterine development in rats. A total of eighty virgin female Sprague-Dawley rats were fed a control diet, a high-fat diet (HF), a high-fat and high-fibre diet (HFF) or a high-fat NAC diet until day 19·5 of gestation. Maternal HFF consumption resulted in a significantly higher mean fetal number and placental weight than in the other groups (P < 0·05). The HFF diet significantly abrogated HF-induced decreases in maternal serum and placental superoxide anion and hydroxyl radical scavenging capacities (P < 0·05); partially abrogated HF-induced increases in maternal serum and placental malondialdehyde (MDA) and protein carbonyl concentrations (maternal serum MDA and placental protein carbonyl, P < 0·05); resulted in significantly higher fetal liver total superoxide dismutase (SOD), Cu- and Zn-containing SOD and Mn-containing SOD (Mn-SOD) activities than in the HF group (P < 0·05). Furthermore, mRNA expressions of hypoxia-inducible factor 1-α, thioredoxin 2 and Mn-SOD in fetal liver and Mn-SOD in fetal heart and placental GLUT3 in the HFF group were higher than those in the other groups (P < 0·05). The inclusion of dietary fibre in the HF diet was more effective than NAC supplementation in maintaining maternal serum and placental superoxide anion and hydroxyl radical scavenging capacities close to those of the control. These results suggest that maternal fibre intake during pregnancy is beneficial for fetal intrauterine development possibly through the improvement of maternal, placental and fetal antioxidant capacities and placental nutrient transfer capacity.

Adolescent girl with unexplained intellectual disability in who was identified a defect in the biosynthesis of creatine by guanidinoacetate methyltransferase (GAMT) deficiency. Were identified two GAMT gene mutations: c.59G>C in exon 1 and c.521G>A in exon 5. The creatine and ornitine substitution and arginine restriction, despite initiated at 13 years old, motivated a global behaviour comportmental acceleration. The GAMT deficiency is an inborn error of metabolism with molecular and clinical heterogeneity varying from a speech delay to autistic behaviour and severe cognitive deficit. Should be considered in patients with unexplained intellec tual disability, especially in Portugal where was documented a high frequency of patients and carriers. The pertinence of the neonatal screen in our country is discussed.

Cerebral creatine deficiency disorders that result in very low levels of creatine in the brain, can cause in intellectual disability, seizures, expressive speech disorder and behavior disorders if not treated in early childhood. CCDDs comprise disorders of creatine synthesis (arginine:glycine [AGAT; MIM 602360]; guanidinoacetate methyltransferase deficiency [GAMT; MIM 601240]) and of creatine transport (SLC6A8 deficiency [SLC6A8; MIM 300036]). Inborn errors of creatine synthesis-but not, as yet, of transport-can be treated by creatine substitution and are thus treatable causes of intellectual disability.

Guanidino compounds in guanidinoacetate methyltransferase deficiency, a new inborn error of creatine synthesis

Dissociation of AGAT, GAMT and SLC6A8 in CNS: Relevance to creatine deficiency syndromes

Paradoxical reactions (PRs) are poorly studied complex immunological phenomena, among patients with tuberculosis (TB). When PRs involves critical structures like the central nervous system (CNS), immunomodulatory therapy is often required. Predictors for PRs in TB to pre-empt appropriate treatment strategies in high-risk groups are lacking. TT genotype of Leukotriene A4 hydrolase (LTA4H) promoter region rs17525495 polymorphisms are associated with exaggerated immune responses in Tuberculous meningitis (TBM), the most severe form of extrapulmonary tuberculosis (EPTB). The association of these polymorphisms with PRs is not known. We evaluated this plausibility among 113 patients with EPTB, at high risk of PRs. Majority [81 (71.7%)] had disseminated tuberculosis with prominent CNS [54 (47.8%)] and lymph node involvement [47 (41.6%)]. Human immunodeficiency Virus (HIV) co-infection was seen among 23 (20.3%) patients. PRs were noted in 38.9% patients, at a median duration of 3 months (IQR 2–4). LTA4H rs17525495 single nucleotide polymorphism (SNP) analysis showed 52 (46%) patients had CC, 43 (38.1%) had CT and 18 (15.9%) had TT genotypes. There was no statistically significant difference in occurrence [CC 38.5% vs CT 39.5% vs TT 38.7%] and time of onset [median (IQR)] of PRs across the genotypes [CC 3 (1–4.7), CT 3 (2–5), TT 2 (2–3)]. PRs was shown to be significantly linked with HIV co-infection (RR 0.6, 95% CI 0.29–1.28), culture positivity (RR 0.5, 95% CI 0.28–1.14), TB Lymphadenitis (RR 0.7, 95% CI 0.44–1.19) and CNS involvement RR 2.1, 95% CI 1.27–3.49) in the univariate analysis (p < 0.2). On multivariate analysis, CNS involvement alone was associated with PRs (aRR 3.8 (1.38–10.92); p < 0.01). PRs were associated with CNS involvement but not with LTA4H rs17525495 polymorphisms.

The effects of 24 hours of nitrous oxide exposure on reproductive indices and fetal development were examined in Sprague-Dawley rats. Four different experiments employing four concentrations of nitrous oxide--0.75%, 7.5%, 25% and 75%--established that the threshold of toxicity was greater than 25%. At 75% nitrous oxide there was a significant increase in early and late resorptions, and a consistent teratogenic effect (e.g., runts, ocular malformations, limb deformities). Neither the stress of shipping dams while pregnant nor the withholding of food during nitrous oxide exposure resulted in additional adverse effects. Exposure to 25% nitrous oxide was associated with increased deoxyuridine suppression values; however, adverse reproductive effects were not seen at this nitrous oxide concentration. The results of this and other studies which have examined the reproductive and teratogenic effects of nitrous oxide do not contraindicate its use in operating rooms nor, when necessary, as an anesthetic for pregnant surgical patients.

Therapeutic trial of arginine restriction in creatine deficiency syndrome.

Guanidinoacetate methyltransferase (GAMT) deficiency is an autosomal recessive inborn error of creatine synthesis that results in intellectual disability, epilepsy, expressive language delay, and dystonia. We report data from two siblings with an uncommon GAMT deficiency phenotype and their clinical, biochemical, imaging, and treatment findings. The older sibling had intellectual disability, epilepsy, and generalized dystonia. The younger sibling had intellectual disability and generalized dystonia. After treatment with creatine, verbal fluency improved, as well as dystonia and aggression. This study confirms that in patients with unexplained intellectual disability, epilepsy, and/or movement disorders, GAMT deficiency should be considered. GAMT-associated cerebral creatine deficiency syndrome is a potentially treatable condition and can be identified by elevated levels of guanidinoacetate in plasma or urine or by a significantly decreased creatine peak on magnetic resonance spectroscopy.