Chapter 142. Disorders of Creatine and Ornithine Metabolism

Creatine is synthesized mainly in the liver and pancreas by the action of arginine:glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT) with arginine, glycine, and S-adenosylmethionine as essential substrates (eFig. 142.1). AGAT catalyzes the first of the two reactions involved in the de novo synthesis of creatine. This reaction utilizes arginine and glycine as substrates and yields guanidinoacetate and ornithine as products. Guanidinoacetate is further converted to creatine by the action of GAMT, using S-adenosylmethionine as a methyl group donor. Creatine reaches muscle and brain via an active transmembrane creatine transport system (CRTR). Creatine is then utilized in the cellular pool of creatine/creatine-phosphate, which together with creatine kinase and ATP/ADP provides a high energy phosphate buffering system. Intracellular creatine and creatine phosphate are nonenzymatically converted to creatinine, with a 1.5% constant daily turnover rate of body creatine. Creatinine is excreted in urine, and the daily urinary creatinine excretion is directly proportional to total body creatine.

Creatine deficiency syndromes represent a group of inborn errors of metabolism, including disorders of creatine synthesis (AGAT; OMIM 602360) and GAMT (OMOM 601240)[ deficiency, and disorders of creatine transport, including the X-linked transmembrane creatine transporter (X-CRTR; MIM 300036) deficiency.1 Inheritance of GAMT and AGAT deficiency is autosomal recessive, whereas the gene for X-CRTR deficiency (SLC6A8) is located on the X chromosome. GAMT and AGAT deficiency seem to be rare disorders, whereas X-CRTR deficiency has been diagnosed in up to 2% of patients who have X-linked mental retardation. This frequency is comparable to the frequency of fragile X syndrome, which constitutes a rather frequent cause of X-linked mental retardation.

Clinical Presentation

The main clinical symptoms observed in all three creatine deficiency syndromes are mental retardation with pronounced speech delay, autistic behavior, and seizures. Five patients from only two families have been described with AGAT deficiency so far. The presenting features included delayed psychomotor and language development and occasional seizures. Patients with GAMT deficiency may exhibit a more complex clinical phenotype, including intractable epilepsy, extrapyramidal movement disorder, and abnormal signal intensities of the basal ganglia.2 The clinical phenotype of X-CRTR deficiency varies from mild to severe mental retardation associated with speech delay and epileptic seizures. Dysmorphic features, microcephaly, and moderate brain atrophy have been described in some of these patients. Heterozygote females may have learning difficulties. Skewed X-inactivation may cause pronounced clinical manifestations in these cases similar to the male phenotype. Interestingly, patients with disorders of creatine synthesis and creatine transport do not have signs of cardiomyopathy or pronounced signs of skeletal myopathy, although muscle tissue seems to be another site of creatine depletion.

Pathophysiology

Patients with disorders of creatine synthesis have systemic depletion of creatine and creatine phosphate due to impairment of de novo creatine biosynthesis. Patients with X-CRTR deficiency have an intact de novo synthesis of creatine, but due to impairment of transmembrane creatine transport, they have intracellular creatine and creatine phosphate depletion, while extracellular (urinary) creatine concentrations are normal or even elevated. The brain is the major site of creatine depletion in all these disorders. Patients with GAMT deficiency have accumulation of guanidinoacetate in addition to creatine deficiency. As guanidinoacetate is neurotoxic in high concentrations, this compound is presumed to play a major role in the pathophysiology of GAMT deficiency.

Diagnosis

The typical biochemical abnormality of creatine deficiency syndromes is cerebral creatine deficiency, which is demonstrated by in vivo proton magnetic resonance spectroscopy. Creatine has a prominent proton magnetic spectrum in the brain, and its deficiency cannot be overlooked (eFig. 142.2).

Guanidinoacetate accumulates in GAMT deficiency and is deficient in AGAT deficiency. Thus, measurement of guanidinoacetate in body fluids may discriminate GAMT deficiency (high concentration) from AGAT deficiency (low concentration). In patients with X-CRTR deficiency, the urinary creatine excretion relative to the creatinine excretion is elevated, and an elevated urinary-creatine-to-creatinine ratio can be used as a first biochemical diagnostic marker for this disease. The diagnosis of all these disorders is confirmed by molecular genetic analysis of the respective genes and by studies of enzyme activities and creatine uptake, respectively. In particular, GAMT and AGAT deficiency is confirmed enzymatically by determining the respective enzyme activities in fibroblasts or virus-transformed lymphoblasts. X-CRTR deficiency is confirmed by creatine uptake studies in cultured fibroblasts.

Treatment3

Cerebral creatine deficiency, as caused by disorders of creatine synthesis (GAMT and AGAT deficiency), can be corrected by oral supplementation of creatine monohydrate (400 mg/kg body weight/day). Treatment of GAMT deficiency also requires therapeutic measures to reduce the accumulation of guanidinoacetate. This is mainly achieved by dietary restriction of arginine, which is the rate-limiting substrate to the synthesis of guanidinoacetate. Substitution of ornithine, which competitively inhibits AGAT activity and thus reduces the synthesis of guanidinoacetate, may enhance this effect. Treatment leads to substantial clinical benefit, including significant developmental progress in AGAT deficiency and improvement of epilepsy and movement disorder in GAMT deficiency. Early recognition and treatment in the newborn period might prevent major symptoms and signs of both disorders. Unfortunately, in X-CRTR deficiency, oral creatine substitution does not result in an increase of brain creatine levels, and no alternative causal treatment is available for this disorder. (For an overview of clinical, biochemical, and diagnostic features and treatment of creatine deficiency syndromes, see eTable 142.1.)

Ornithine is a nonprotein amino acid that has various functions (eFig. 142.1). First, it is a key intermediate in the urea cycle. There, it is produced from arginine via the arginase reaction, yielding ornithine and urea. This reaction occurs in the cytoplasm. Second, ornithine is transported via the ornithine transporter (ORNT1 or SLC25A15 gene) into the mitochondrium, where it serves as an essential substrate for ornithine transcarbamylase (OTC) in the intramitochondrial part of the urea cycle. OTC catalyzes the formation of citrulline from ornithine and carbamyl phosphate. Third, intramitochondrial ornithine is also substrate to ornithine aminotransferase (OAT), which catalyzes the conversion of ornithine to Δ1-pyrroline-5-carboxylate (P5C), which is an essential substrate for proline synthesis. Fourth, extramitochondrial ornithine is the key substrate for the formation of polyamines, spermine, and spermidine, which are involved in DNA packaging. Fifth, ornithine is derived from a reaction that arginine undergoes with glycine in order to form guanidinoacetate, an essential intermediate in creatine synthesis. This reaction is catalyzed by arginine amidinotransferase (AGAT), and ornithine plays a regulatory role (via allosteric inhibition) in the activity of this enzyme.4

Three disorders affecting ornithine metabolism are known in humans.5 (1) ornithine aminotransferase (OAT) deficiency, which causes gyrate atrophy of the choroid and retina (MIM 258870); (2) ornithine transporter (ORNT1 or SLC25A15 gene) deficiency, which causes hyperornithemia-hyperammonemia-homocitrullinuria (HHH) syndrome (OMIM 238970). The third defect is a newly described disorder in the synthesis of ornithine and proline (P5C) synthase deficiency, which catalyzes an essential step in the synthesis of proline and ornithine from glutamate. While OAT deficiency and HHH syndrome are characterized by hyperornithinemia, P5C deficiency is characterized by hypoornithinemia.

OAT deficiency is a rare autosomal recessive disorder with a relatively high prevalence in Finland. Clinically, OAT deficiency is characterized by ocular manifestations: patients experience night blindness in early childhood as a first sign of retinopathy. Other ocular findings include myopia and posterior subcapsular cataract. As retinopathy progresses, patients lose peripheral vision, develop a tunnel vision, and become virtually blind in the adult age. Muscle weakness, EEG changes, brain atrophy, and peripheral nervous system involvement have been described as additional complications in single patients.

Pathophysiologically, OAT catalyzes the conversion of ornithine to Δ1-pyrroline-5-carboxylate (P5C). Normally the reaction equilibrium is directed toward the production of P5C. Thus, in OAT deficiency, hyperornithinemia is the biochemical hallmark. Urinary excretion of ornithine is also high, and due to competitive inhibition of tubular reabsorption, excretion of lysine, arginine, and cystine may also be elevated. Hyperornithinemia results in inhibition of creatine synthesis. Therefore, patients with OAT have moderately decreased creatine levels in the muscles and brain. It is not known whether low creatine levels contribute to the pathophysiology of the disease. Interestingly, in neonates the reaction equilibrium of OAT is directed conversely, namely toward synthesis of ornithine from P5C. Thus, neonates with OAT deficiency have low levels of ornithine rather than hyperornithinemia. Lack of ornithine results is depletion of substrates for the urea cycle and thus results in low levels of citrulline and arginine and in hyperammonemia.

Diagnosis

OAT is diagnosed by demonstration of high plasma ornithine levels, which typically are 5 to 20 times higher than the normal range. Diagnosis is confirmed by measuring OAT activity in fibroblasts and by mutational analysis of the OAT gene.

Treatment

Treatment aims to correct the amino acid abnormalities. Pyridoxal phosphate is a cofactor of OAT, and some patients respond to high dosages of pyridoxine with fair correction of the ornithine levels. In nonresponders, hyperornithinemia is partially corrected by dietary arginine restriction through a low-protein diet supplemented with arginine-free amino acid mixtures. The goal of treatment is to slow the progression of retinopathy. The effects of this treatment on the long-term outcome of retinopathy and other disease manifestations are not yet known. As neonates with OAT deficiency have decreased levels of ornithine and arginine, an arginine-restricted diet is contraindicated in this period of life.

This is a rare syndrome of hyperammonemia, hyperornithinemia and homocitrullinemia; it is autosomal recessive disorder of ornithine transport across the mitochondrial membrane (ORNT1, SLC25A15 gene).

Clinical Presentation

The clinical picture is mainly characterized by hyperammonemia, which results from intramitochondrial ornithine depletion and thus insufficient supply of ornithine as substrate for the urea cycle. Due to nutritional protein loads and catabolism, patients develop intermittent hyperammonemia associated with vomiting, ataxia, lethargy, irritability, confusion, and other psychiatric manifestations. Neonatal onset and lethal hyperammonemic coma have been described in single cases. In the interim, patients are asymptomatic. In the long term, patients may develop chronic neurological deficits (eg, progressive spastic paraparesis), developmental delay, and mental retardation. Although extramitochondrial ornithine is high, patients are not likely to develop retinopathy as one would expect from hyperornithemia caused by OAT deficiency.

In addition to hyperornithinemia and hyperammonemia, the disorder is characterized by homocitrullinuria, which arises from alternative metabolism of carbamyl phosphate via lysine. Orotic aciduria, which is a common feature of various urea cycle defects, is also present in HHH syndrome as a sign of carbamyl phosphate accumulation.

Diagnosis

Diagnosis of HHH syndrome is established by the characteristic combination of elevated ornithine levels in blood and elevated excretion of homocitrulline in urine along with intermittent hyperammonemia. Confirmation of the diagnosis is possible by ornithine incorporation assays in fibroblasts and by mutational analysis of the SLC25A15 gene.

Treatment

Treatment is directed toward preventing increased ammonia production through a protein-restricted diet, avoidance of catabolic states . Citrullin supplementation helps to reduce the ammonia load, and it consumes additional nitrogen via its conversion to arginine.

Only two patients from one family have been described with P5C synthase deficiency.4,6 These patients’ clinical signs were indicative of involvement of both the nervous system and the connective tissue. Particular manifestations included muscular hypotonia, frequent vomiting, failure to thrive, cutis laxa, hyperextensible joints, developmental delay, seizures in infancy, cataracts, progressive spasticity, and severe intellectual deficit later in life. As P5C synthase acts at the interface between ornithine and proline synthesis, both hypoprolinemia and hypoornithemia are characteristic diagnostic features. As a consequence of hypoprolinemia, citrulline and arginine are low as well, and hyperammonemia may occur as a result of insufficient supply of these substrates to the urea cycle. Treatment with ornithine and proline has not been effective in preventing the clinical manifestations. (For an overview of the clinical, biochemical, and diagnostic features and treatment of disorders affecting ornithine metabolism, see eTable 142.2.)