An Introduction into Citrullinemia, an Autosomal Recessive Disease

Introduction

Citrullinemia is a type of amino acid disorder caused by an inherited deficiency of specific enzymes which prevents ammonia build-up (Nitrogen accumulation in the form of ammonia) in the body. It is one of the Urea Cycle Disorders (UCD). There are 2 types of Citrullinemia, each causing different signs and symtoms and are contributed by mutations in different genes.

Type I affects 1 in 57,000 people worldwide, therefore being the most common type of the disorder. Type II citrullinemia affects about 1 in 100,000 to 230,000 individuals in Japan. This form of disorder is also seen in other parts of the world, including people from East Asia and the Middle East.

The Urea Cycle

This cycle takes place in the liver, where excess nitrogen produced by protein metabolism is being processed to urea. Urea is then excreted by the body...

In mitochondria:

1. Ammonium+Hydrogen carbonate+2ATP--CPS1-->carbomyl phosphate+2ADP+Pi
2. Ornithine+Carbomyl phosphate--OTC-->Citrulline+Pi

In cytosol:

1. Citrulline+Aspartate+ATP--ASS1-->Arginosuccinate+AMP+PPi
2. Arginosuccinate--ASL-->Arginine+fumarate
3. Arginine+H20--ARG1-->Ornithine+Urea

Till date, there are no other alternative pathways known to be able to synthesize urea. Therefore, any disruption of any step in the urea cycle would be fatal as accumulation of ammonia levels are toxic to us. Non-funtional/partially functional enzymes, involved in Urea Cycle, derived from mutation of certain genes, could cause blockage of the reactions. This leads to increased levels of ammonia in the blood (hyperammonemia).

Type 1 (classic): mostly diagnosed in infants and children

 

Neonatal acute form

Infant appears normal at birth, however, after a few days, infant starts to develop the symptoms. About 56% of patients are symptomatic by 4 days old, about 67% develop symptoms after a week old.

Late onset form

Clinical course of late onset form could be milder than or the same as the acute neonatal form, however only occurs at a later stage of life. Though symptoms and effects of hyperammonemia may be similar to the neonatal onset, the neurologic findings are more subtle due to older age of patients.

Roots of CTLN1

Caused by mutation in the ASS1 gene (located on chromosome 9), which is responsible for producing the enzyme argininosuccinate synthetase 1 (ASS1). ASS1 synthesizes arginosuccinate from citrulline and aspartate (amino acids).

Mutation of the gene decreases the enzymatic ability to metabolise nitrogen effectively.

Absence of arginosuccinate prevents the cycle from progressing and prevents formation of arginine, which is then converted to urea. Therefore, ammonia and other harmful substances gets built up in the blood. Along with that, levels of citrulline is also elevated. Without this enzyme, formation of Urea is interrupted and excess (waste) nitrogen is prevented from elimination.

The mutation of ASS 1 gene is due to the substitution of one amino acid, altering the structure of the gene. This affects the ability of ASS1 to bind to citrulline and other molecules. The defected ASS1 disrupts the processing of excess nitrogen, in the form of ammonia, into urea.

This enzymatic deficiency is found in all tissues that express ASS.

Type 2: also diagnosed in adults (adult-onset)

Roots of CTLN2
Caused by mutation in SLC25A13 (Solute Carrier Family 25, member 13) gene, which is responsible for making the protein citrin. Gene is located on chromosome 7.


Within liver cells, Citrin transports glutamate into the mitochondria, and transports aspartate out (malate-aspartate shuttle). In order for aspartate to enter the urea cycle, sufficient amount of aspartate have to be transported out. Mutation in the SLC25A13 gene prevents transportation of aspartate into cytosol, and therefore prevents formation of arginosuccinate. This leads to discontinuation of the Urea cycle, and contributes to improper metabolism of hamful nitrogen molecules.

There is a decreased level of ASS1 enzyme in liver cells, however, the enzyme concentrations in other tissues/cells are normal.



Effects of Ammonia Accumulation

Citrullinemia can cause

• muscle weakness
• poor growth
• enlarged livers
• mental retardation
• respiratory problems (difficulty breathing)
• Brain damage (swelling)
• Paralysis, death

Signs and Symptoms

Type 1
Symptoms become evident during first few days of life (infant)

• Lethargy
• Poor-feeding
• Seizures
• Unconsciousness

A milder form of the disease can develop in late childhood/adulthood. Symptoms are as follows:

• Intense headaches
• Loss of vision (partial)
• Ataxia (lack of muscle and balance coordination)
• Lethargy

Type 2
Symptoms caused by type 2 citrullinemia may appear during adulthood

• Confusion
• Aggression
• Restlessness
• Memory loss
• Hyperactivity

*These symptoms can be triggered by medications, alcohol intake, and infections.



Diagnosis

Patients with extreme citrullinemia have plasma ammonia concentration of 1000-3000 µmol/L. The Plasma Quantitative amino acid analysis reports an absence of arginosuccinic acid and high concentration of citrulline (greater than 1000 µmol/L).

*Normal range of citrulline is 50 µmol/L.

There is also a decrease in ASS1 enzymatic activity in  in fibroblasts, liver, and other tissues where the enzyme is expressed. Molecular genetic testing of ASS1 gene is clinically available.

Prenatal diagnosis can be performed by measuring activity of ASS enzyme in cultured chorionic tissue and amniocytes.

Treatment
The treatment focuses on the prevention of ammonia build-up


1) Low-protein diet. This diet should continue throughout the patient's lifetime.

• A diet with high levels of protein causes accumulation (build-up) of ammonia in the body due to increased level of amino acid derived from protein molecules. But, this does not mean protein should be 100% eliminated from the child's diet. Small amounts of protein can be consumed in carefully measured amounts, to support the growth and development of the child.

2) Medication. Used to eliminate ammonia in the body.

• Arginine ( an amino acid), is used as a supplement to aid ammonia removal. It is one of the by-products of the Urea cycle, and therefore allows Urea cycle to be completed. *Refer to diagram of Urea cycle*

• In the event of high/inclination of ammonia level in the blood, medications are given intravenously, or a dialysis is performed to regulate the blood ammonia level.
  

*Regular blood tests must be taken to monitor ammonia and amino acid levels.

 

 

Prevention of Citrullinemia

Since it is a genetic disease obtained via inheritance, there is no ultimate way to prevent Citrullinemia. However, there are certain methods used to minimize the occurance of the disorder.


Prenatal Diagnosis

• DNA or enzyme testing using Chorionic Villus Sampling (CVS) or amniocentesis can be done, whereby the amniotic fluid is obtained.

• Above is an example of the results collected for an enzyme test. It is seen that the levels of citrulline in the various fetal tissues are elevated. However, the levels of other amino acids were significantly lesser than the controls tested.

Carrier Detection (before pregnancy)

• It is important to undergo genetic counseling for adults who are carriers/affected, to discuss the potential risks of the offspring being affected with the disorder.