Glycine encephalopathy

Glycine encephalopathy (GE), also known as non-ketotic hyperglycinemia or commonly just NKH, is a rare, inherited, autosomal recessive disorder of glycine metabolism. The condition arises from defects in the glycine cleavage system, an essential enzyme for glycine break down. This results in toxic accumulation, particularly in the brain causing seizures, lethargy, feeding difficulties, respiratory problems, intellectual disabilities, and a high risk of early death.There are several forms of the disease, with varying severity of symptoms and time of onset. The diagnosis of NKH is often initially suspected based on abnormally high levels of the amino acid glycine in bodily fluids and tissues, especially the cerebrospinal fluid. After phenylketonuria, glycine encephalopathy is the second most common disorder of amino acid metabolism.

Glycine encephalopathy is often referred to as "non-ketotic hyperglycinemia", a reference to the biochemical findings seen in patients with the disorder, and to distinguish it from the disorders that cause "ketotic hyperglycinemia" (as seen in propionic acidemia and several other inherited metabolic disorders). To avoid confusion, the term "glycine encephalopathy" is sometimes used, as the term more accurately describes the clinical underpinnings of the disorder.

The first signs and symptoms usually appear in the neonatal period (first hours or days of life) and to a lesser extent the infantile period (2 weeks to 3 months). For onset in the neonatal period, the hallmark sign is progressive lethargy often leading to coma and strong hypotonia (muscle weakness). If not supported by ventilation death often occurs. Most regain spontaneous respiration and see improvement in alertness in the first month of life including bottle drinking. Additionally, pronounced hiccups or myoclonic jerks may be present, which are themselves often a sign of epilepsy.^[1] If onset occurs in the infantile period, the primary symptom is protracted hypotonia combined with developmental delays and seizures. Although late presentation (beyond 3 months) is possible, it is uncommon and typically features a more subtle presentation of symptoms, such as developmental delays and mild seizures. Infants with this presentation are frequently associated with the attenuated form of the disease.^[2][3]

In infants who survive and are managed with sodium benzoate, a range of symptoms may manifest, dependent upon the severity of the disease. The most prominent symptom is seizures which are present in a majority of cases. Other common symptoms include continued hypotonia and lethargy. In the attenuated forms, these symptoms are often milder than the severe form. Seizures are readily controlled and individuals can learn to walk, reach, grasp, and may learn to speak or use sign language. Lethargy is often occasional and linked to infections. Additionally, hyperactivity is common and often severe and treatment resistant. In the severe form, however, seizures are difficult to treat and may become progressively worse. Children do not learn to sit or grasp and often have limited ability to interact with their environment. They also often develop cortical blindness, scoliosis, and hip dysplasia. There is typically no hyperactivity, but spasticity is common. Some have cleft palate or clubfeet and secondary microencephaly has also been noted.^[2][4]

Glycine encephalopathy has an estimated incidence of 1 in 60,000, making it the second most common disorder of amino acid metabolism, after phenylketonuria. It is caused by a defect in the glycine cleavage system (GCS), which is made up of four protein subunits. Each of these four subunits is encoded by a separate gene. Defects in three of these four genes have been linked to glycine encephalopathy.^[5]: 790

There is a fourth unit in the GCS: dihydrolipoamide dehydrogenase or GCSL. However, to date there have been no mutations in GCSL found to be associated with glycine encephalopathy.

A small percentage of affected individuals do not have detectable mutations in any of the three genes (listed above) that are typically associated with the disease. However, they still show defective glycine-cleavage enzymatic activity. This is called variant NKH, where patients have mutations in the genes encoding one of the cofactors associated with the GCS complex, such as defects in the lipoate synthesis or iron-sulphur cluster biogenesis .^[6]

Defects in the GCS proteins can prevent the complex from functioning properly or can prevent the GCS complex from forming entirely. When the complex is unable to metabolize glycine properly, this causes excess glycine to build up to toxic levels in the body's organs and tissues. Damage caused by elevated levels of glycine in the brain and cerebrospinal fluid is responsible for the characteristic seizures, breathing difficulties, movement disorders, and intellectual disability.

This disorder is inherited in an autosomal recessive pattern. The term "autosomal" signifies that the gene associated with the disorder is located on an autosome. In an autosomal recessive inheritance pattern, two defective copies of the gene (one inherited from each parent) are required in order for a child to be born with the disorder. Therefore, each parent of an individual with an autosomal recessive disorder has at least one defective copy of the gene. With autosomal recessive disorders, individuals with only one copy of a defective gene (heterozygotes) are considered "carriers" for the disorder. Carriers usually do not show signs or symptoms of the disorder.^[7]

Glycine is the simplest amino acid, having no stereoisomers. It can act as a neurotransmitter in the brain, act as an inhibitor in the spinal cord and brain stem, while having excitatory effects in the cortex of the brain. Glycine is metabolized to final end products of ammonia and carbon dioxide through the glycine cleavage system (GCS), an enzyme complex made up of four protein subunits. Defects in these subunits can cause glycine encephalopathy, although some causes of the disease are still unknown. Normally, GCS shows its highest enzymatic activity in liver, brain and placental tissue. One of its main functions is to maintain normal glycine levels in the brain. Defects in GCS cause an increase of glycine concentration in blood plasma and cerebrospinal fluid.^[5] The exact pathophysiology of the disorder is not known, but it is considered likely that buildup of glycine in the brain is responsible for the symptoms.^[8]

All forms of glycine encephalopathy show elevated levels of glycine in the plasma, as well as in cerebral spinal fluid (CSF).^[5]: 793 Glycine concentrations in the CSF of affected patients are typically more markedly elevated than in plasma, leading to a corresponding elevation in the ratio of glycine concentrations in the cerebral spinal fluid to that in the plasma. This ratio can also be slightly elevated in patients receiving valproic acid.^[5]: 811

Glycine encephalopathy (nonketotic hyperglycinemia, or NKH) should not be confused with other metabolic disorders that can produce elevated glycine levels. For example, certain inherited 'organic acidurias' (aka 'organic acidemias') can produce elevated glycine in plasma and urine, although these disorders are not caused by defects in the glycine cleavage system, and they are not typically accompanied by corresponding elevations of glycine in cerebrospinal fluid (CSF).^[9] Glycine encephalopathy is unique in the fact that levels of glycine are disproportionately elevated in CSF (in addition to elevations in blood and urine), whereas CSF glycine levels are normal or near-normal in patients with inherited organic acidurias.

Glycine is metabolized in the body's cells to end products of carbon dioxide and ammonia. The glycine cleavage system, which is responsible for glycine metabolism in the mitochondria is made up of four protein subunits, the P-protein, H-protein, T-protein and L-protein.^[5]: 793

There are several different forms of glycine encephalopathy, which can be distinguished by the age of onset, as well as the types and severity of symptoms. All forms of glycine encephalopathy present with only neurological symptoms, including intellectual disability (IQ scores below 20 are common^[10]), hypotonia, apneic seizures, and brain malformations.^[5]: 811

With the classical, or neonatal presentation of glycine encephalopathy, the infant is born after an unremarkable pregnancy, but presents with lethargy, hypotonia, apneic seizures and myoclonic jerks, which can progress to apnea requiring artificial ventilation, and often death. Apneic patients can regain spontaneous respiration in their second to third week of life. After recovery from the initial episode, patients have intractable seizures and profound intellectual disability, remaining developmentally delayed. Some mothers comment retrospectively that they noticed fetal rhythmic "hiccuping" episodes during pregnancy, most likely reflecting seizure episodes in utero.^[8] Patients with the infantile form of glycine encephalopathy do not show lethargy and coma in the neonatal period, but often have a history of hypotonia. They often have seizures, which can range in severity and responsiveness to treatment, and they are typically developmentally delayed.^[11] Glycine encephalopathy can also present as a milder form with episodic seizures, ataxia, movement disorders, and gaze palsy during febrile illness. These patients are also developmentally delayed, to varying degrees. In the later onset form, patients typically have normal intellectual function, but present with spastic diplegia and optic atrophy.^[11] The mild form of the disorder corresponds to greatly reduced but not fully absent GCS activity.^[12]

Transient neonatal hyperglycinemia has been described in a very small number of cases. Initially, these patients present with the same symptoms and laboratory results that are seen in the classical presentation. However, levels of glycine in plasma and cerebrospinal fluid typically normalize within eight weeks, and in five of six cases there were no neurological issues detected at follow-up times up to thirteen years. A single patient was severely retarded at nine months. The suspected cause of transient neonatal hyperglycinemia is attributed to low activity of the glycine cleavage system in the immature brain and liver of the neonate.^[8]

There is no cure for NKH and no treatments that alter the natural history of the disease.^[13] The food and cosmetic preservative, sodium benzoate, is used to manage symptoms primarily by normalizing plasma glycine levels.^[14] This occurs through a two-step reaction. Sodium benzoate breaks down into benzoic acid in the body which is converted to benzoyl-CoA via benzoyl-CoA synthetase in the mitochondria of the liver and kidneys. Benzoyl-CoA then combines with glycine via the enzyme glycine N-acyltransferase to form hippuric acid (hippurate) which is excreted through the urine.^[15][16] Sodium benzoate has a variable effect on brain glycine levels and cannot normalize cerebrospinal fluid glycine levels.^[17][18] In the United States, sodium benzoate is not a stand-alone FDA approved treatment for any condition and patient’s must source it themselves and at their own risk.^[19]  

NMDA receptor antagonists, including dextromethorphan or ketamine, function by inhibiting the N-methyl-D-aspartate receptors for which glycine serves as a co-agonist and thus may be over stimulated in NKH. These therapies primarily aid with seizure control and may improve neurodevelopmental outcomes in some individuals.^[12] Still, many patients often need three to four seizure medications for adequate control.^[20]

The ketogenic diet is frequently employed alongside the previously mentioned therapies. As a therapeutic diet, it is known to effectively address difficult-to-control seizures in children.^[21] Furthermore, this diet enables patients to reduce their sodium benzoate dosage, likely owing to its promotion of gluconeogenesis, which utilizes glycine and other glucogenic amino acids to generate glucose.^[22][23]

The prognosis is very poor. Two studies reported typical age of deaths in infancy or early childhood, with the first reporting a median age of death of 2.6 for boys and less than 1 month for girls.^[24][10] Despite this, reports exist of select individuals living into their 50s.^[25]

Gene therapy seeks to replace the defective gene responsible for the glycine cleavage system with a functional copy. Current research has predominantly utilized AAV9, an adenoviral vector capable of penetrating the brain, to deliver a working copy of the GLDC gene in neonatal mice. These studies have shown a reduction in both plasma and brain glycine levels, along with a normalization of the folate profile, indicating a restoration of the glycine-derived one-carbon supply.^[26][27]

As of now, there are no approved systemically delivered gene therapies that exist to treat disorders primarily affecting the brain. However, onasemnogene abeparvovec has been approved for spinal muscular atrophy, a condition affecting the spinal cord and certain brain regions. AAV9's ability to cross the blood-brain barrier is present at any age, but maximal efficacy is observed in neonates.

Early research combined sodium benzoate with acetylsalicylic acid (commonly known as aspirin). Salicylic acid and glycine combine to form salicyluric acid primarily in the kidneys and is the main way salicylates are excreted. No research has thoroughly investigated its usage in NKH. Other research has investigated sodium cinnamate as alternative glycine lowering therapy and shown its effectiveness.^[28] Notably, sodium benzoate is a metabolite of cinnamon and likely works to lower glycine in a similar fashion. No therapeutic strategies have focused on lowering brain glycine levels.

The glycine receptor is one the most common inhibitory receptors in the brain and plays key roles in motor conotrol, pain, respiration, and brain development. Research in the late 1970s to mid-1990s explored using strychnine, a glycine receptor antagonist, to treat NKH with no benefit. Strychnine is highly toxic and no longer used in medicine.

Intractable seizures in NKH are thought to arise from overstimulation of NMDA receptors by glycine, which functions as a co-agonist of the receptor. While NMDA receptor antagonists like dextromethorphan and ketamine have shown to be helpful in some patients they do not function by preventing glycine binding of the receptor. Several safe drugs that prevent glycine binding on the NMDA receptor have been developed, such as L-4-chlorokynurenine, Gavestinel, and Licostinel, but did not prove to be better than placebo in their respective trails for depression and stroke. None have been tested in NKH.

Metabolism of glycine is a major provider of one carbon carrying folates which are depleted in NKH. This has shown to be associated with neural tube defects, prenatal aqueduct stenosis and subsequent hydrocephalus in GLDC mutant mice which can be prevented by supplementation of formate, an alternative source of 1-carbon donors. Moreover, a study in 1974 by Tribjels and colleagues showed that sodium formate was able to reduce glycine levels, but not normalize them in an individual with NKH.

Response to treatment is variable and the long-term and functional outcomes of both the disorder and its management remain largely unstudied.To provide a basis for improving the understanding of the epidemiology, genotype/phenotype correlation and outcome of these diseases and their impact on the quality of life of patients, and for evaluating diagnostic and therapeutic strategies a patient registry was established by the noncommercial International Working Group on Neurotransmitter Related Disorders (iNTD).^[29] Additionally, a patient registry was established with the Coordination of Rare Diseases at Sanford (CoRDS) and overseen by the patient advocacy organization NKH Crusaders^[30]

NKH is a rare disease and ultimately ultra-rare disease with an estimated incidence of 1 in 76,000 globally.^[31] The highest known incidence is potentially Finland at 1 in 12,000.^[32]

The physician Barton Childs and his team published the first case of what they called 'ketotic hyperglycinemia' in 1961. Their patient displayed elevated levels of ketones and glycine and was the first description of what is now known more accurately as propionic acidemia.^[33] In 1965, four years later, Gerritsen and colleagues described 'a new type of idiopathic hyperglycinemia' of elevated glycine levels without elevated ketones becoming the first description of 'non-ketotic hyperglycinemia'.^[34]

NKH is a rare disease, but maintains a very active global community presence from educating parents with newly diagnosed children to fundraising for treatments. Organizations and groups in this space include the following in alphabetical order: Brodyn's Friends, Drake Rayden Foundation, Jack Richard Urban Foundation, John Thomas NKH Foundation, Joseph's Goal, Les Petits Bourdons, Lucas John Foundation, Maud & Vic Foundation, Nora Jane Foundation, NKH Crusaders, NKH Network, The Foundation of Non-Ketotic Hyperglycinemia, The Mikaere Foundation.

• List of amino acid metabolism disorders
• inborn errors of metabolism
• GLYT1 encephalopathy