Genetics of Hyperammonemia-Hyperornithinemia-Homocitrullinuria (HHH) Syndrome

Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome, also called ornithine translocase deficiency, is a very rare inborn error of metabolism; the age at presentation and long-term prognosis widely vary among affected individuals. Growth and developmental delays, learning disabilities (especially speech delay), and periodic confusion and ataxia are typical presenting symptoms. In this syndrome, a defect in the transport of ornithine into the mitochondrial matrix significantly inhibits the urea cycle, thereby impeding nitrogen disposal. Early detection and treatment may lead to favorable outcome.

The urea cycle maintains the concentration of the toxic ammonium ion in a narrow, tolerable range despite a 10-fold variation in the dietary intake of its precursor, nitrogen. A total of 5 enzymes in 2 subcellular compartments (mitochondrial matrix and cytosol) convert ammonia into urea, which is excreted by the kidney (see image below).

Periportal hepatocytes express these enzymes; epithelial cells of the small intestine and kidney also express these enzymes to a lesser extent, but their contribution to urea production is not significant. Urea-cycle enzyme activity is regulated by dietary protein. In part, glucagon and cyclic adenosine 3',5'-monophosphate (cAMP) regulate urea-cycle enzyme transcription.

The first 2 steps of the urea cycle occur in the mitochondrial matrix. Carbamoyl phosphate is produced from ammonia and bicarbonate by carbamoylphosphate synthetase I. This reaction is stimulated by ornithine. An inner mitochondrial membrane transporter directs ornithine to the transcarbamoylase enzyme to keep intramatrix ornithine levels low. The specifics of the liver transporter have recently been identified.

Cationic L-ornithine is electroneutrally transported into the matrix in exchange for a proton and citrulline. The inner membrane pH gradient and the availability of proton-yielding anions may affect the transport rate. As with other mitochondrial carrier family proteins, the ornithine carrier is composed of 300 amino acids that constitute 3 repeated motifs of approximately 100 amino acids each. These motifs contain 2 hydrophobic alpha-helical segments connected by an extensive hydrophilic sequence, resulting in 6 transmembrane portions of the protein.

The transporter was identified by probing a mammalian-expressed sequence tag database with 2 fungal mitochondrial ornithine carrier protein sequences. Ornithine incorporation was restored in fibroblasts derived from patients with hyperornithinemia-hyperammonemia-homocitrullinuria syndrome by transforming the fibroblasts with transporter complementary DNA (cDNA). Incorporation was traced using ornithine labeled with radioactive carbon (14C).

Following incorporation of ornithine into the mitochondrial matrix, carbamoyl phosphate and ornithine are condensed to form citrulline by ornithine transcarbamoylase. Citrulline is believed to passively diffuse across the inner mitochondrial matrix to the cytosol. The contribution of the ornithine/citrulline antiporter to citrulline transport from the mitochondria to the cytosol is not known.

The next 3 steps of the urea cycle occur in the cytosol. Argininosuccinic acid is produced from the condensation of citrulline and aspartate by a synthetase enzyme. It is then cleaved to produce fumarate and arginine by a lyase enzyme. Urea and ornithine are produced by arginase. Under normal circumstances, the ornithine produced outside the mitochondrial matrix is transported into the mitochondrial matrix, where it is reused in the urea cycle.

This transport of ornithine across the inner mitochondrial membrane is essential to the urea cycle. Ornithine can also be produced in the matrix by aminotransferase, but this enzyme is active in pericentral venous hepatocytes rather than in periportal hepatocytes.

In hyperornithinemia-hyperammonemia-homocitrullinuria syndrome, the mitochondrial ornithine transporter ORNT1 is defective. The carrier protein and gene sequence have only recently been identified; before its identification, the carrier's dysfunction was deduced biochemically because a patient with hyperornithinemia-hyperammonemia-homocitrullinuria syndrome has abnormally high ornithine levels despite normal ornithine transcarbamoylase function. Because the urea cycle cannot continue without ornithine inside the mitochondria, ammonia disposal slows, and blood ammonia levels rise. A second mitochondrial ornithine transporter, ORNT2, has been suggested and may account for a mild variation of hyperornithinemia-hyperammonemia-homocitrullinuria syndrome in French-Canadian probands. In some individuals, a gain in ORNT2 transporter function may compensate for the ORNT1 deficit.

Ornithine transcarbamoylase within the mitochondrial matrix may convert lysine to homocitrulline in the absence of ornithine, causing high blood levels of homocitrulline and homocitrullinuria. However, this theory is controversial because some studies have shown no correlation between lysine supplementation and homocitrulline levels; moreover, the role of the lysine transcarbamoylase that lies outside the inner mitochondrial membrane is not known.

Several recent studies suggest that ornithine, homocitrulline, and ammonia accumulation results in damage to the brain through increased reactive species and mitochondrial dysfunction.[1, 2, 3, 4]

 

Frequency

International

Only about 50 cases have been reported.

Mortality/Morbidity

Neonatal death has been reported but is rare. Some patients have progressive neurologic and cognitive deterioration, whereas other patients demonstrate good function if metabolic anomalies are well controlled. Clearly, this is a very serious disorder that is potentially life-threatening and often life-shortening.

Race

Most reported cases have been in the French-Canadian population in the Quebec Province of Canada.

Sex

The male-to-female ratio is unknown.

Age

The severity ranges from minimal neurologic dysfunction in adulthood to neonatal death. Age at diagnosis also widely varies, probably, in part, because of variation in the degree of residual enzyme activity and because of the nonspecific symptoms of this disorder.

Growth improves with treatment.

Pregnancy is possible. A woman with hyperornithinemia-hyperammonemia-homocitrullinuria syndrome gave birth to a healthy baby on a diet that consisted of 1 g/kg of protein per day.

Some patients do not respond to diet therapy.

One case of rapidly progressive deterioration after formula feeding, leading to neonatal death, has been reported.

Older patients may develop progressive disease, but patients who have had good neurologic and cognitive function into adulthood have been reported. Abnormalities in the central and peripheral nervous systems in older patients can be detected using electrophysiologic tests (see Other Tests). The following manifestations can also occur:

• Irritability, opposition, and aggressiveness, suggesting a conduct disorder
• Lower IQ score (ie, in the mildly mentally retarded range)

Clotting factors VII and X may be deficient in patients with hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome.

A sibling with the disorder or consanguinity is not uncommon.

Ask about a history of previous neonatal deaths or miscarriages.

Common presenting signs include the following:

• Developmental delays

• School difficulties

• Recurrent liver dysfunction

• Increased levels of transaminases with mild coagulopathy detected on laboratory tests

• Episodic lethargy and vomiting may be presenting signs.

The history varies depending on age of onset, as follows:

Neonatal onset

Vomiting and lethargy following feeding of high-protein formula suggests formula intolerance.

The neonatal period may be uneventful if the neonate is breastfed.

Symptoms may be mild and may include only bottle refusal.

Severe hyperammonemia with rapidly progressive deterioration after formula feeding is rare but has occurred.

Infant onset

Symptoms may coincide with the introduction of high-protein solid food around the time of weaning.

Choreoathetosis episodes may occur, with normal neurological function between episodes.

Hypotonia may progress to spasticity.

Seizures may resemble infantile spasms.

Developmental milestones are typically delayed.

Growth may be retarded.

Childhood onset

Ataxia or choreoathetosis episodes may occur, with normal neurological function between episodes.

The child may refuse to eat meat and fish or to drink milk.

Other signs may include seizures, developmental delays, polyneuropathy, episodic confusion, gait disturbance, learning disabilities, a below-average intelligent quotient (IQ) score, attention deficit hyperactivity disorder (ADHD), conduct disorder, and failure to thrive.

Strokelike episodes may occur.[5]

Liver failure may occur.[5, 6]

Adult onset

Patients may experience learning disabilities.

Patients may avoid high-protein foods and possibly have a vegetarian diet.

Periodic blurred vision, confusion, and ataxia are common symptoms.

Eyes

Retinal depigmentation and chorioretinal thinning are uncommon findings. In contrast, chorioretinal atrophy with punched-out lesions is a standard finding in patients with gyrate atrophy.

Abdomen

The liver and spleen may be enlarged.

Neurologic

See the list below:

• Pyramidal syndrome characterized by increased deep tendon reflexes, spasticity, positive Babinski reflex, and nonpersistent clonus

• Decreased vibration sensation

• Buccofaciolingual dyspraxia

• Poor visuomotor function

• Poor hand coordination

• Poor fine-motor coordination

• Dysdiadochokinesia

Development

See the list below:

• Global motor delay

• Speech delay

Hyperornithinemia-hyperammonemia-homocitrullinuria syndrome is a genetic/metabolic disorder caused by a defect in the mitochondrial ornithine transporter ORNT1.

The ORNT1 gene has been mapped to band 13q14. This gene is also identified as SLC25A15 because of its membership in the solute mitochondrial carrier protein family.[7] Its expression is similar to that of other urea-cycle enzyme genes; it is expressed at high levels in hepatocytes, and an increase in dietary protein can promote its expression.

Three ORNT1 mutant alleles were identified in a survey of 11 hyperornithinemia-hyperammonemia-homocitrullinuria probands; these mutant alleles accounted for 21 of 22 possible mutant ORNT1 genes in the population.[8]

In individuals of French-Canadian descent with hyperornithinemia-hyperammonemia-homocitrullinuria, a 3-base-pair (bp) in-frame deletion of codon 188 for phenylalanine, which causes an unstable carrier protein, is common. Ten patients were tested for this mutation; 9 were homozygous, and one was heterozygous. The mutation responsible for the dysfunction of the heterozygote's remaining allele was not identified.

A missense mutation at codon 189, resulting from a G → A transition at bp 538, impaired carrier activity without affecting targeting or stability in a non–French-Canadian patient. The patient was heterozygous for this mutation and had a microdeletion on chromosome 13 that, presumably, accounted for dysfunction in the corresponding allele.

Genotyping studies have repeatedly confirmed that genotype has a poor correlation with phenotype.

Inheritance is autosomal recessive.

Although the genes for clotting factors VII and X are also located on chromosome arm 13q, these genes are believed to be too distant from the ornithine transporter gene to be part of a contiguous gene syndrome.

Amino acid studies reveal the following:

• Plasma ornithine is increased at the time of presentation, which differentiates hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome from other urea-cycle disorders. Ornithine levels may range from 200-1000 µmol/L, slightly lower than in patients with gyrate atrophy. The plasma ornithine level may be lowered by protein restriction or even normalized by extreme protein restriction. Neonatal ornithine levels may be normal.

• Postprandial homocitrullinuria biochemically differentiates this disorder from gyrate atrophy.

• Homocitrulline levels are elevated in the urine. A recently described liquid chromatography tandem mass spectrometric method may be more accurate than older coelution methods.

• Free ornithine levels are elevated in the urine, although they can widely vary. Ornithine metabolite levels and other gamma-glutamyl amino acid metabolite levels may be elevated in urine.

• Glutamine and alanine levels are often elevated at the time of presentation, and glutamine levels may paradoxically increase with protein restriction.

Orotic acid levels in the urine are elevated despite normal serum ammonia values.

Ammonia levels at the time of diagnosis have ranged from 60-216 μ g/dL.

Postprandial hyperammonemia differentiates this disorder from gyrate atrophy.

Random levels are within the reference range if treatment is successful.

Even with treatment, plasma ammonia levels may increase after protein ingestion.

High-protein diets result in chronic hyperammonemia.

Increased levels of liver transaminases and alkaline phosphatase with normal levels of gamma-glutamyl transpeptidase and bilirubin are common.

Increased lactic acid levels and an elevated lactate-to-pyruvate ratio have been reported.

Lactate and Krebs cycle intermediates can be found in the urine.

Coagulation factors VII and X should be measured and may be deficient.

Cultured skin fibroblasts from patients with hyperornithinemia-hyperammonemia-homocitrullinuria syndrome or ornithine aminotransferase deficiency incorporate only one sixth the amount of labeled tracer ornithine into protein as control fibroblasts.

In this test, cells are incubated with [14C]ornithine and leucine labeled with tritium. The labeled leucine provides a measure of general protein synthesis.

In fibroblasts, ornithine is not used in the urea cycle but is processed in the mitochondrial matrix to form glutamate, which is subsequently incorporated into proteins.

The ratio of 14C to tritium incorporated into cellular protein is measured.

The amount of 14C incorporated into fibroblasts from patients with hyperornithinemia-hyperammonemia-homocitrullinuria syndrome is typically only 15% of that incorporated into control fibroblasts.

This test has been extremely useful in the diagnosis of hyperornithinemia-hyperammonemia-homocitrullinuria syndrome.

MRI may reveal increased signal in cortical white matter, subcortical or cortical atrophy, or basal ganglia calcifications; conversely, the findings may be normal.

Liver-spleen scan may reveal increased uptake with mild diffuse liver involvement.

Electrophysiologic studies may reveal abnormalities in older patients. Findings may include the following:

• Electroencephalogram that reveals diffuse slowing of background activity

• Nerve-conduction velocity and short-latency somatosensory–evoked potential results compatible with mild sensorimotor peripheral neuropathy

• Visual-evoked potential results revealing prolonged cortical conduction time and shape and amplitude anomalies

Liver biopsy reveals distended vacuolated periportal hepatocytes filled with intracytoplasmic and intranuclear glycogen.

Nuclei are small and contain dense chromatin.

The rough endoplasmic reticulum is decreased. The smooth endoplasmic reticulum is highly developed, giving it a stacked appearance.

Mitochondria in hepatocytes, myocytes, leukocytes, and fibroblasts may be large and bizarre in shape and size, with segmented ridges, lamellar crystal-like inclusions, and innumerable closely packed and parallel cristae.

Ornithine supplementation reduces ammonia levels in some patients with hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome. A suggested dose of 22-44 mg/kg per dose administered 3 times per day with protein ingestion may improve protein tolerance and growth. Other studies show that 6 g/d reduces ammonia levels. This treatment further increases ornithine levels, and the long-term effects of hyperornithinemia are not known. Citrulline supplementation has also been used.

Arginine supplementation (7.5 g/d) reduces ammonia levels in some patients; however, this treatment has caused deleterious effects in others and is generally not recommended.

Sodium benzoate and sodium phenylacetate may reduce ammonia levels by providing an alternative pathway. A combination of sodium benzoate and sodium phenylacetate (Ammonul) is an intravenous drug for use in urea-cycle disorders. Oral sodium phenylbutyrate, which has been approved by the US Food and Drug Administration (FDA) for urea-cycle defects, could be helpful in hyperornithinemia-hyperammonemia-homocitrullinuria syndrome. However, additional studies are needed. Oral sodium benzoate could also be effective.

Glycerol phenylbutyrate is a pre-prodrug that undergoes metabolism to form phenylacetate. Results of a phase 3 study comparing ammonia control in adults showed glycerol phenylbutyrate was noninferior to sodium phenylbutyrate.[9] In a separate study involving young children ages 2 months through 5 years, glycerol phenylbutyrate resulted in a more evenly distributed urinary output of PAGN over 24 hours and accounted for fewer symptoms from accumulation of phenylacetate.[10]

Hyperammonemic crisis might be managed with short-term protein restriction and intravenous fluids that contain large amounts of glucose, followed by slow reintroduction of small amounts of protein. Theoretically, intravenous arginine and intravenous sodium benzoate and sodium phenylacetate might be effective, but these medications have not been approved in the United States for use in this disorder, and intravenous arginine could be dangerous and ineffective. Supportive measures are indicated.

If the high blood ammonia levels are due to the reduction of N-acetylglutamate or to the reduction of carbamoyl-phosphate synthase-I activity, N-carbamylglutamate (carglumic acid) can be administered together with the conventional therapy.[11]

A comprehensive team approach is justified and should include a metabolic disease specialist, a clinical biochemical geneticist, a developmental pediatrician, a neurologist, and other development specialists. This team should assess all aspects of cognitive function and periodically monitor the patient for development surveillance.

A nutritionist with expertise in treating metabolic diseases should also be consulted.

A low-protein diet (1.2 g/kg/d, depending on age) may prevent postprandial hyperammonemia and has permitted normal development in several patients when initiated early in life.

Hyperornithinemia-hyperammonemia-homocitrullinuria syndrome may be diagnosed in first trimester by studying the incorporation of [14C]ornithine into proteins of chorionic villi cells. Although this is theoretically possible, it has never been reported in the literature.

Amniocytes demonstrate decreased incorporation of [14C]ornithine, but amniotic fluid amino acid levels are normal.

Because neonatal ornithine levels may be normal, especially if the patient is asymptomatic, these levels cannot be used to screen neonates for this disorder.

Annual ophthalmologic examinations and electroretinography are often recommended in patients with hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome.

Growth and developmental milestones need to be closely monitored.

Follow-up should be approached with a comprehensive development team.

A dietary log helps to track total daily protein ingestion.

Ammonia, liver transaminases, and ornithine levels should be periodically monitored, especially after changing or starting diet or supplement therapy.

Sodium benzoate and sodium phenylacetate may reduce ammonia levels by providing an alternative pathway.

Class Summary

These agents assist in excreting nitrogen and serve as an alternative to urea in reducing waste nitrogen levels. Administer only in a large medical facility with close laboratory monitoring available.

Sodium phenylacetate and sodium benzoate (Ammonul, Ucephan)

Benzoate combines with glycine to form hippurate, which is excreted in urine. One mol of benzoate removes 1 mol of nitrogen. Phenylacetate conjugates (via acetylation) glutamine in the liver and kidneys to form phenylacetylglutamine, which is excreted by the kidneys. The nitrogen content of phenylacetylglutamine per mole is identical to that of urea (2 mol of nitrogen). Ammonul must be administered with arginine for carbamyl phosphate synthetase, ornithine transcarbamylase, argininosuccinate synthetase, or argininosuccinate lyase deficiencies. Indicated as adjunctive treatment of acute hyperammonemia associated with encephalopathy caused by urea-cycle enzyme deficiencies. Serves as an alternative to urea to reduce waste nitrogen levels.

Glycerol phenylbutyrate (Ravicti)

Glycerol phenylbutyrate is a nitrogen-binding agent for chronic management of adult and pediatric patients (including newborns) with urea cycle disorders who cannot be managed by dietary protein restriction and/or amino acid supplementation alone. It is a pre-prodrug that is metabolized by ester hydrolysis and pancreatic lipases to phenylbutyrate and then by beta oxidation to phenylacetate. Glutamine is conjugated with phenylacetate to form phenylacetylglutamine, a nitrogen waste product that is excreted in the urine. It is not indicated for treatment of hyperammonemia.