Research article

LIVING-RELATED LIVER TRANSPLANTATION (LRLT) FOR TYPE II

CITRULLINEMIA USING A GRAFT FROM

HETEROZYGOTE DONOR

Introduction

Citrullinemia is an autosomal recessive disease which prevents proper metabolism of amino acids into urea, resulting in excess ammonia accumulation in blood which, could lead to death. Type II Citrullinemia is distinguished by absence of liver-specific argininosuccinate synthetase (ASS) due to mutation of SLC25A13 gene. The main objective of liver transplantation is to provide the body with the enzyme responsible for amino acid metabolism. Nine patients received transplantation from relatives that are heterozygote carriers who had normal metabolic parameters. The measured enzymatic activity of ASS in liver of donors was in the normal range, but genotyping of the affected gene was not tested because the affected gene and its mutation were distinguished after the operation. LRLT is a fundamental method in liver transplantation due to the scarcity of organs from dead bodies (cadaveric donors). Therefore, there might be occurrence of risks by the usage of graft from heterozygote donors. This report covers the transplantation of liver graft from a heterozygote father.

Method

The 16 years old male patient showed his first symptoms of vomiting and drowsiness, with high serum ammonia level. The elevation of plasma citrulline and arginine level, and level of serum pancreatic secretory trypsin inhibitor (PSTI) served evidence as the onset of citrullinemia. Despite medications, several episodes of hyperammonemia and liver disfunction arose, resulting in need of plasma exchange. Patient’s serum ammonia level decreased and his conscious level improved after treatment. He was later diagnosed with Citrullinemia Type 2 after detection of mutated SLC25A13 gene and study of plasma amino acid pattern. Analysis of plasma amino acid concentration of parents showed normal levels of citrulline and arginine. Results for gene analysis showed his parents being asymptomatic heterozygote while the patient is a homozygote for IVS1111G.A mutation. His father was selected as a donor due to a better graft-to-recipient weight ratio. The transplantation was performed using the left lobe of his father. During the anhepatic period, a portosystemic shunt was done between the infrahepatic inferior vena cava and the right portal branch to restrain from portal hypertension. Microscopic examination of postoperative native liver specimen revealed only 1.5% of control ASS activity, as compared to the graft liver with normal activity of five urea cycle enzymes involving ASS.

Results

The postoperative course for both the father and son was natural. After a day, patient’s plasma amino acid concentrations were normalized and citrulline level dropped to a normal range. It took around ten days before patient’s arginine level normalized. The amount of ammonia fluctuates greatly in the first ten days, but slowly reaches the normal range. Patient was released 50 days after the operation when both citrulline and arginine concentrations were stabilized in the normal range.

Conclusion

Having had twelve successful cases, the use of living-related liver transplant could be an acceptable treatment for citrullinemia. However, a series of tests would have to be done to ensure that the donor for the graft is genetically heterozygote. Another method, the auxiliary partial orthotopic liver transplantation (APOLT) was mentioned to be a treatment for acute liver failure. It is said that it could accommodate for the deficiency of enzymes without removing the liver completely. With this, the body is prepared in case of any graft failure in the future. However, the risk of rejection with APOLT is very severe to the extent of an irreversible brain damage. Therefore, performing LRLT with the use of a graft from a genetically proven heterozygote donor was chosen instead.

Word Count: 573 words

Reference(s)

Midwest's First Liver Cell Transplant for Baby with Life-Threatening Urea Cycle Disorder Performed at Children's Memorial, WEINHEIM, Germany, Dec. 13, 2011 /PRNewswire/ Retrieved from http://www.prnewswire.com/news-releases/midwests-first-liver-cell-transplant-for-baby-with-life-threatening-urea-cycle-disorder-performed-at-childrens-memorial-135509483.html

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.

Case Study

Midwest's First Liver Cell Transplant for Baby with Life-Threatening Urea Cycle Disorder Performed at Children's Memorial




Introduction

Considering the severity of the disease, accumulation of neurotoxic ammonia leads to severe nerve and brain damage. Untreated children experience abnormal physical and mental development. Liver transplantation seems to be the sole cure, but is an exceptionally tedious procedure for neonatal and very young patients.

A clinical trial, which was approved by the U.S Food and Drug Administration in 2010, was held to evaluate the efficacy and safety of liver cell transplantation in patients 5 years old and below. The trial involves enrolling 20 patients across 14 centers in United States of America and Canada, following an analysis of interim results based on an ongoing trial in Germany, which involves newborns with UCD.

Midwest's Baby

The baby who was diagnosed with Citrullinemia received an investigational liver cell therapy sponsored by International Biotechnology firm Cytonet. The transplantation was done at the Children's Memorial Hospital in Chicago.

The baby is now undergoing follow-up care at Rady Children's Hospital, San Diego.

Brief steps in liver transplantation

1. Collection of healthy cells from donated livers not suitable for transplantation
2. Gentle isolation of liver cells, complex processing of cells
3. Infusion of cells into portal veins over 6 days

*Risk of portal thrombosis and systemic shunting of liver cells

Potal thrombosis (PVT)

• Portal vein is a large vein in the abdomen which carries blood from the bowel and other abdominal organs to the liver. PVT occurs when the vein gets blocked with blood clot, causing blood to back up in the vein and leading to high pressures in the vein below it.

Systemic shunting

•  Systemic shunting occurs when blood from the abdominal organs bypasses the liver and is instead sent to the systemic circulation. Therefore, toxins and nutrients are not filtered/absorbed.  

Thoughts of the wise...

"A challenge is enrolling patients since UCD only affects about 400 babies born in the United States each year and diagnosis poses several challenges." Dr. Wolfgang Rudinger, CEO and CSO of Cytonet.

"Unfortunately, because of the rarity of this disease, it's non-specific symptoms and because newborn screening panels do not catch all cases, many babies with severe forms of the disease are not diagnosed or the diagnosis comes too late." Bruce Barshop, M.D., Ph.D., professor of pediatrics and co-director of the Biochemical Genetics Laboratory at the University of California.

"We are pleased to be participating in this trial because it has the potential to provide a treatment option for children with a rare and complicated diagnosis for which there are few options now," said Barbara K. Burton, MD, a pediatrician and director of the PKU program at Children's Memorial.

Reference: Midwest's First Liver Cell Transplant for Baby with Life-Threatening Urea Cycle Disorder Performed at Children's Memorial, WEINHEIM, Germany, Dec. 13, 2011 /PRNewswire/

Link:http://www.prnewswire.com/news-releases/midwests-first-liver-cell-transplant-for-baby-with-life-threatening-urea-cycle-disorder-performed-at-childrens-memorial-135509483.html

Do ponder...

What an interesting discovery! The human body is indeed a complex mechanism, which nature had established. Just think on how different molecules and structures within our organs coordinate together to perform such intricate and complicated process, how slight mutations in our genes could lead to massive disruption to the peaceful cycle nature has set for this machine (body). *Just a minute, do you remember about a scientist wanting to create new life forms out of protein molecules? Do you think he's able to regenerate organisms that are utterly, speechlessly complex as humans?*

Anyway...

Let's all hope and pray that the post-liver transplantation recovery period goes well for the Midwestern Baby. And let's all pray towards advancements and improvements in the quality of life, with the aid of scientific discoveries. *Well unless scientists dont misuse those discoveries and start having absurd ideas, like producing sheeps without their fur. This could harm the eco system! What were they thinking?*


TOODLES~

References

Content/Images:

http://ghr.nlm.nih.gov/condition/citrullinemia

http://ghr.nlm.nih.gov/gene/ASS1

http://www.newbornscreening.info/Parents/aminoaciddisorders/ASAS.html

http://www.ncbi.nlm.nih.gov/books/NBK1458/

http://www.chem.uwec.edu/Chem454_S09/example1.pdf

http://www.elmhurst.edu/~chm/vchembook/633ureacycle.html

http://www.ajnr.org/content/23/2/334.full