Argininemia - Arginase Deficiency from Mutation of Arg1 Gene
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WAGNER, D. -. (2000). Argininemia - Arginase Deficiency from Mutation of Arg1 Gene. National Undergraduate Research Clearinghouse, 3. Available online at Retrieved April 25, 2017 .

Argininemia - Arginase Deficiency from Mutation of Arg1 Gene

Sponsored by: TODD ECKDAHL (
ABSTRACT Argininemia is a rare, recessive disorder of the urea cycle. The ARG1 gene expresses itself in human liver tissues and synthesizes the arginase enzyme, the last step of the urea cycle. Patients that cannot manufacture this enzyme build up ammonia in their blood and can suffer from mental retardation and ultimately may die. Worldwide there are known to be only 28 individuals that suffer from this disorder. It`s rarity is most likely due to misdiagnoses and being undiagnosed preceding death. Reye`s syndrome is often a misdiagnosis of this disease. There are 14 known mutations of the ARG1 gene that can cause arginase deficiency. It is believed that 20% of all Sudden-Infant-Death-Syndrome fatalities may be attributed to undiagnosed urea cycle disorders. Current gene therapy trials have shown promising results for urea cycle disorders and could be beneficial to someday treating this disease.

Argininemia is a rare, hereditary, urea cycle disorder. It is included in the category of inborn metabolic disorders of which there are no cures. It is also referenced in the literature as hyperargininemia, ARG1 deficiency, and arginase deficiency. This genetic disease is characterized by the absence of the enzyme, arginase. Arginase is the last enzyme necessary in the urea cycle, which is a metabolic pathway occurring in the liver. The metabolism of proteins in the diet creates nitrogen, as a waste product, in the circulatory system. The urea cycle processes this nitrogen, in the form of ammonia, from the blood, through a sequence of biochemical processes. Arginase is responsible for the incorporation of ammonia into urea, which can then be excreted from the body in urine. If urea cannot be synthesized, nitrogen accumulates in the blood and body tissues, as ammonia, which is a highly toxic substance, and may lead to brain damage and /or death. Arginase is also the enzyme necessary to regenerate the urea cycle, converting arginine to ornithine and beginning the cycle again. Argininemia is a rare, inborn, metabolic disorder. A study of 28 individuals, in 1992, (Grody, 1992) claimed to represent most arginase deficient persons, worldwide. Disorders of the urea cycle are often misdiagnosed or lead to brain damage and/or death before a proper diagnosis can be made. It is believed that 20% of Sudden Infant Death Syndrome infants may succumb to one of the inborn metabolism disorders, such as a urea cycle disorder, that is undiagnosed, (NUCDF). Argininemia presents itself as an autosomal, recessive, heterogeneous disorder. Of the cases studied, there does not appear to be a correlation between individuals born of consanguineous or non-consanguineous parents. Occurrences are distributed worldwide, with cases identified in persons of the following ethnicities: French-Canadian, Spanish, Puerto Rican, Mexican, Pakistani, Portuguese, Cambodian, German, Saudi-Arabian, and Australian. A large proportion of the research done in this disorder has originated from Japan, so there are more references to the frequency of this disorder within the Japanese population, than in the other groups. The definitive symptoms of argininemia include elevated arginine levels in the blood, urine, and cerebro-spinal fluid. Red cells are low in arginase activity. The enzymatic properties (but not enzymatic activities) of arginase are identical in fetal and adult red cells, providing a diagnostic testing method to be used for pre-natal testing via amniocentesis. Diagnostic testing can be done by immunoprecipitation-competition and Western blot to attempt to locate the arginine1 protein. Numerous other symptoms occur which do not lead to a direct arginase deficiency diagnosis, but rather are often representative of mis-diagnoses. Rising blood-ammonia levels, hyperammonemia, are responsible for the myriad of symptoms that are possible. Argininemia does not cause as severe an increase in the blood-ammonia levels as do the other urea cycle disorders. Any of the following symptoms may present itself at any stage of the disorder, they are not inclusive and/or age-specific. Infants may appear irritable, vomit, and become lethargic. This may be followed by seizures, poor muscle tone (hypotonia), respiratory distress, coma and eventual death, if untreated. Small children may be hyperactive, sometimes exhibiting self-injurious behaviors, and screaming. They may refuse to eat meat and other high protein foods. It is common for these children to be referred to child psychologists for behavioral and eating disorders. Some early childhood viral illnesses, such as chickenpox, may bring on episodes of hyperammonemia, as can high-protein meals, and exhaustion. These conditions have been misdiagnosed in children as Reye`s Syndrome. It was once believed that children with urea cycle disorders did not survive to adulthood. In recent years there appears to be an alarming increase in the numbers of adults that are being diagnosed with urea cycle metabolic disorders. Adults may exhibit stroke-like symptoms, lethargy, and delirium. They are often referred to psychiatrists based on these behaviors. These symptoms may be preceded by a viral illness, childbirth, or precipitated by the use of anti-seizure medicines. In addition to the symptoms, consequences of an arginase deficiency can lead to retardation, mental impairment, and instances of microcephalaly (abnormally small cranial capacity). Paralysis, partial and total, is a common effect. This can include spastic episodes, ataxia (inability to control voluntary muscular movements), lower limb weakness, and hypotonia. Cerebral palsy is often the misdiagnosis for these symptoms. Individuals may be protein intolerant and can have episodes of seizures, convulsions, or collapse. Episodes of hepatomegaly, enlargement of the liver, can cause tenderness or become quite severe. If misdiagnosed or left untreated, eventual coma and death will result. Although there are no cures for urea cycle disorders, there are several treatment options that are clinically in use. For the less severe cases of argininemia, a strict dietary restriction of low protein consumption can be beneficial. This lowered protein intake limits the amounts of available nitrogen to be converted in the urea cycle. The essential amino acids are often administered orally, to supplement the diet, as are vitamins and calcium supplements. Medications are available that can provide alternative pathways for removing ammonia from circulation but these are unpalatable and administered by either a stomach or nasal tube. One treatment entailed the oral administration of the amino acid lysine to compete with arginine, as they are chemically similar, but this did not result in the desired effect. Liver transplants have also been used to treat this disorder, with mixed results. It has been discovered that researchers that had been working with the Slope virus had low levels of arginine in their blood. The Slope virus is a DNA virus that has been used in clinical testing of gene therapy trials. By inoculating the Slope virus into fibroblasts from tissue cultures of argininemic individuals, they have monitored an increase in arginase activity. This may be a therapy avenue that can lead to future treatment of this disease. Recent advances in gene therapy have been made with a similar urea cycle disorder, ornithine transcarbamylase deficiency. This therapy was proving to be successful with the individuals in the trial. Unfortunately, the human trials have been suspended due to the death of 18 year old Jesse Gelsinger in September 1999, he was the last individual being treated in Phase 1 of the trials. Future gene therapy trials have currently been halted, pending the thorough investigation of this unfortunate event.

The gene responsible for the production of arginase is the ARG1 gene located at the 6q23 loci on the human chromosome 6. This gene is 11.5 kilobases long. The gene has 8 exons, several which have been identified as playing a role in the occurrence of argininemia. A "TATA" box sequence is located 28 base pairs upstream from the cap site, which was determined by nuclease S1 mapping and primer extension. A sequence similar to the binding sites of the transcription factor, "CAAT" box, is located 72 bases upstream. The ARG1 gene is responsible for transcribing the protein arginase. Arginase catalyzes L-arginine + H2O = L-ornithine + urea. This catalysis occurs within the liver, where arginase is cytoplasmic. Mutations of the gene occur as a variety of point mutations, not as a structural gene deletion. Argininemia is transmitted as a rare, Mendelian, autosomal, recessive disease. The severity of the disease is contingent on the types of molecular defects in the arginase gene. Nine nucleotide (missense/nonsense) substitutions, two splicing nucleotide substitutions, and three small deletions had been identified as of 1990 by Haraguchi ( As of 1995, Uchino, identified 21 of 22 mutant alleles involved in 9 mutations, that exhibited varying degrees of disease severity and response to treatment. In the early 1980`s, ARG1 and ARG2 had not been separated. A rabbit antibody to the human-liver arginase (ARG1) was used to examine the immunologic characteristics of arginase in red blood cells, liver, kidney, brain, and the gastrointestinal tract. The liver arginase was precipitated at 90%; whereas the kidney, brain, and GI arginase only precipitated at 50%. It was found that the diseased patients had arginase in their red blood cells that was immunoreactive to the antibody but inactive enzymatically. With the use of immunodiffusion and precipitation-inhibition studies it was demonstrated that there were two types of human arginase proteins and therefore; two gene loci in humans. ARG2 has since been pinpointed to the mitochondria of the kidneys. It can be induced, and its levels elevated by as much as 4X, in the absence of ARG1. It is believed that this increase in ARG2 is responsible for ureagenesis. Mutation studies were done by Southern blots of genomic DNA, which was cut with restriction enzymes and probed with a near full-length human-liver-arginase cDNA clone. Rat liver ARG1 cDNA, was used to probe human-liver derived cDNA. A clone of the human gene was used in human-rodent cell hybrids to assign the gene to chromosome 6. By using a combination of somatic cell hybrid analysis and in-situ hybridization with the human DNA probe (1,550 bp) for the gene, they were able to map the gene to the 6q23 location. Three individuals studied had a loss of the Taq1 cleavage site. This was traced by electrophoretic analysis of the PCR amplified fragment, after it was treated with Taq1, and confirmed by sequencing. The loss of the Taq1 site was caused by base substitutions in exon 8, codons 290 and 291. Arginine 291 was changed to a terminator and Thr 290 was changed to Ser, adjacent to the Taq1 site. Frameshift deletions cause two mutations; 4 bases deleted in the protein coding regions 262-265 or 263-266, in exon 3, shift the frame after amino acid 87 and create a new stop codon at residue 132, and a 1 base deletion at nucleotide 77 or 78 in exon 2, causes a frameshift after residue 26 and creates a stop codon at residue 31. A GA mutation at nucleotide 365 converts tryptophan 122 to a stop codon. The mutated protein that forms has 1% of its normal activity and is not stable. A GC mutation at nucleotide 235 in exon 7 changes Gly235 to Arg, the protein formed also has only 1% of its normal activity. Deletion of nucleotide 842 of cDNA, cytosine, in exon 8 converts leucine282 to tryptophan, this in turn causes a frameshift and creates a stop codon at 289, the protein formed has no activity. A base substitution of TC at nucleotide 32 in exon 1 replaces isoleucine with threonine at codon 11, this protein has 12% normal activity. A base substitution of Gt at nucleotide 413 in exon 4 replaces valine for glycine at codon 138. A splice site mutation at nucleotide 57 causes a substitution that violates the GT/AG rule for splice site junctions. An AG substitution can occur at the acceptor site of intron 4, nucleotide 466. A CT substitution in exon 2 results in Arg21 converting to a terminator.

A mutation of the ARG 1 gene causes a deficiency in the cytosolic liver-type arginase enzyme. This enzyme is 322 amino acid residues long with a molecular mass of 34,732 and is catalogued as L-arginine urea-hydrolase E.C. The human-liver arginase approximates 1450 bp. The ARG1 gene expresses itself in the liver as the ARG2 expresses in the kidney. The high level of expression in the liver has been postulated to be mediated by an enhancer in intron 7 (Vockley), the liver arginase deficiency may be related to a functional deficiency in this enhancer region. There is a 92% conserved identity in the two genes in their six short regions and a 42% conserved identity in the remainder of the sequences. Analysis by Northern blot has shown the ARG1 gene expressed as a 1.4 kb mRNA in the liver, only. The ARG1 enzyme contributes 98 % of the arginase activity in the liver and is absent (with quantitative variation) in cases of argininemia. The ARG 1 gene operates in the cis configuration.

Argininemia can be detected by many means; however, it is often misdiagnosed in clinical settings. Liver tissues do not have any arginase activity; this can be detected by biopsy. Red blood cells and body tissues can be checked for arginase activity, but this is not a reliable method. Blood arginine levels are generally elevated as is the arginine levels of the cerebrospinal fluid. Urinary tests may detect elevated levels of arginine citruline and argininosuccinic acid. Arginase activity is undetectable in amniotic fluid; however, fetal red cells have identical arginase properties to those of adults. It is possible to test for argininemia with these cells obtained by amniocentesis or amnioscopy. The blood may be checked for high ammonia levels; however, argininemia does not consistently present the hyperammonemia associated with the other urea cycle disorders. Western-blot can be preformed using anti-liver arginase antibody cross-reacting with arginase material in red cells. Many treatments have been attempted with varying results. Mortality rates for this disease are estimated at 50%, this is hard to calculate as it is believed that there are many fatalities annually that are not correctly linked to do disorder as the cause of death. Medications are available that will provide alternative pathways for the removal of ammonia from the blood. This are administered via a gastrointestinal tube and are supplemented by amino acid therapy, multiple vitamins, and calcium supplements. Patients with argininemia often require numerous hospitalizations and frequent blood testing. Several liver transplants have been performed as a last resort, but few have been successful. Patients have shown increased sensitivity to the seizure drug, sodium valproate, which is often erroneously prescribed for argininemia sufferers. Oral administration of lysine was tried to enhance dibasic amino acid competition, with poor results. Arginase replacement therapy via erythrocyte transfusion was also tried which did not show any clinical improvement in reducing blood arginine levels. Dietary restrictions are mandatory with protein consumption being carefully monitored or eliminated; this can be supplemented with sodium benzoate treatment. There is believed to be many connections between urea cycle disorders and other major disease such as, cancer, AIDS, and sickle cell anemia. In cancer studies, several of the chemotherapy agents used have caused changes in the urea cycle. Some of the early drugs used for children with urea cycle disorders are being used at major cancer research institutions, appearing to halt production of cancer cells in lymphomas, melanomas, cystic fibrosis and sickle cell anemia. Large population studies have shown correlation between changes in the arginase enzyme and exposure to environmental stresses. There is a therapy avenue being pursued that may use the two separate gene loci for the ARG1 and ARG2 genes to independently manipulate them for therapeutic approaches. Persons working with the Shope virus have been found to have low blood arginine levels. Tissue cultures of argininemic patient`s fibroblasts have been inoculated with the Shope virus and have shown that arginase activity can be induced. It is not known, as yet, how this DNA virus is able to restore arginase activity, but this is a promising avenue of treatment that is being pursued. The most promising treatment for this disease is in gene therapy. Gene therapy trials have been conducted on the urea cycle disorder Ornithine Transcarbamylase. A corrected gene was placed into the body via the recombinant adenovirus transfer, to provide DNA instructions for making the missing enzyme. The results were exceptional, in each of the seventeen patients administered gene therapy, correction occurred. The last patient in Phase 1 of the trials died several days after the therapy and the trials were halted pending further investigation as to the causes of his death. This occurred in November 1999. Phase 2 trials were scheduled to begin by placing the corrected gene in children with OTC. With the promising results for the gene therapy trials for OTC, I believe that it would be feasible to pursue gene therapy for argininemia as well, since both diseases are part of the urea cycle and caused by an enzyme deficiency. The findings on the Phase 1 trials` death have shown that the young man died of causes not directly related to the gene therapy, although they were escalated by it. Hopefully, Phase 2 trials will be able to resume shortly with some protocol changes. There are currently 6 known urea cycles disorders that may benefit from this research and therapy.

BIBLIOGRAPHY National Center for Biotechnology Information -OMIM Online Mendelian Inheritance in Man, OneD- Viewer, Entrez Nucleotide, LocusLink, dbSTS, GeneMap `99, Entrez Genome, PubMed Human Gene Mutation Database at the Institute of Medical Genetics in Cardiff National Urea Cycle Disorders Foundation Frontiers in Clinical Genetics - Real Audio Lecture Series Batshaw, Mark, M.D., Gene Therapy in ChildrenBlaese, Michael R., M.D., Gene Therapy. Where We`ve Been and Where We Are GoingKnoppers, Bartha Maria, LLD, Ethical Issues in GeneticsTuchman, Mendel, M.D., Ornithine Transcarbamylase Deficiency, A Paradigm for X-Linked Disorders IHGT - The Institute for Human Gene Therapy NORD National Organization for Rare Diseases National Institute of Health - Office of Biotechnology Activities------------------------------------------------------------------------

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