Resum's+Disease+Research+Paper

Thomas D. Monteverde Biochemistry CHE 570 Professor Dhar 4 April 2009 __Research Essay: Refsum Syndrome__ Genetic diseases are illnesses caused by abnormalities in genes or chromosomes. Genetic diseases are passed through mutated genes to subsequent generations. In general genetic diseases have a very low incidence rate, occurring yearly in a extremely small percentage of the overall population. The prevalence of genetic diseases (the percentage of the total population affected by a disease) however are much higher, which is due to the chronic and incurable characteristics of most genetic diseases. The prevalence of the diseases being an accumulation of the incidence from successive years. Characteristics and inheritance of genetic diseases are dependent on the genes and chromosomes in which the mutations occurs. A Y-linked genetic disease is caused by mutations in the Y-chromosome which as a result is only inherited by males from their fathers. A X-linked genetic disease is caused by a mutation in the X-chromosome and can be inherited by either son or daughter from either father or mother. Autosomal dominant and recessive genes are non-sex chromosomes and are the same in both sexes. The physiological function for which the gene codes for is commonly affected by its mutation.

Leukodystrophies are a group of genetic diseases which are characterized by the progressive degeneration of the white matter in the brain. These diseases are a result of a mutation in a gene that affects the maintenance and growth of white matter specifically the molecules associated with myelin. Myelin is the insulating sheath that covers the axon of the nerve cells. This sheath enables nerves to conduct impulses from the brain to other parts of the body. One of the eight currently identified hereditary leukodystrophies can prevent the formation of myelin which is Pelizaeus-Merzbacher disease. Pelizaeus-Merzbacher disease is the result of a mutation in the gene that produces the basic protein of central nervous system myelin. The etiology of Alexander's disease is still unkown and the other six leukodystrophies are storage disorders which cause a toxic build up of chemical substances. These toxic chemical substances adversely affect the myelin sheath of nerve cells. Metachromatic leukodystrophy, adrenoluekodystrophy, Krabbe's disease, phenylketonuria, Canavan disease, and Refsum's disease are all storage disorders.

In 1945 Sigvald Bernhard Refsum observed five members of two unrelated Norwegian Families. Sigvald Bernhard Refsum patients were between the ages of twenty five and forty one and all the patients had consanguineous parents of Scandinavian origin. The patients all seemed to suffer from similar symptoms which consisted of atypical retinitis pigmentosa with concentric constrictions of the visual fields; chronic polyneuropathy with symmetrical weakness or paresis of the distal parts of the limbs with absent or diminished deep reflexes; ataxia and nystagmus with other cerebral signs; and increased cerebrospinal fluid protien. Sigvald Bernhard Refsum named the disease heredo-ataxia hemeralopia polyneuritiformis. He later re-named the disease to heredopathia atactica polyneuritiformis due to his inability to prove a link between the disease and familial heredo-ataxia (hereditary incoordination). Heredopathia atactica polyneuritiformis also became known as Refsum's disease. After Refsum's initial findings several other reports of similar cases emerged. Reese and Baretta in 1950; Clark and Critchley in 1951; Austin in 1956; Fleming in 1957; Ashenhurst, Millar, and Milliken in 1958; Heycock and Wilson in 1958; and Gordon and Hudson in 1959 all reported similar cases of heredopathia atactica polyneuritiformis. Despite these reports little more was known about Refsum's disease until nearly ten years after Sigvald Bernhard Refsum's initial study.

Ataxia implies a dysfunction of parts of the nervous system that coordinate movement, such as the cerebellum, but it was doubtful whether ataxia was of cerebellar and spinocerebellar origin in Refsum's disease. Ataxia in combination with tremor and intention tremor is often prominent, but has been shown to be absent in some cases. If ataxia was found in individuals changes relating to or caused by Refsum's disease in parts of the nervous system that coordinate movement were rare and if there were changes they were slight. In 1956 a scientist named Cammermeyer suggested that ataxia is secondary to proprioceptive deafferentation. This would mean that the conduction of impulses are slowed instead of a dysfunction in the receptors. Studies of peripheral nerve conduction velocity have shown marked slowing usually to a degree which is compatible with demyelination in most cases of Refsum's disease. Cammermeyer has also revealed evidence of a process of demyelination and remyelination that causes the formation of round lipid bodies on Schwann cells near the nuclei and lipid crystalline patterns located in mitochondria.

In 1963 W. Kahlke and E. Klenk found a massive accumulation of phytanic acid (3, 7, 3 11, 15 teramethylhexadecanoic acid) in all postmortem tissue from a patient who suffered from Refsum's disease. The accumulation of phytanic acid was found in all tissues and blood samples. The possible connection between concentrations of phytanic acid and Refsum's disease led Kahlke to continue study cadavers that suffered from Refsum's disease. By 1964 Kahlke had demonstrated the presence of phytanic acid in the lipids of at least nine cases of Refsum's disease. Phytanic acid accounted for over fifty percent of fatty acids in the liver and the concentration in the kidneys were almost as great. It was found that phytanic acid levels in plasma of Refsum's disease patients are greater than 200 µmol/L compared with normal levels of less than 30 µmol/L. Another study was preformed by Daniel Steinberg in 1966 where trace doses of phytol-U-14C were orally administered to seven control subjects and two patients with Refsum's disease. Labeled phytanic acid was found in the plasma of all test subjects which shows that phytol in the diet is precursor for phytanic acid. The fraction of the absorbed dose converted to 14CO2 in twelve hours was 3.5% and 5.8% in Refsum's disease patients and averaged 20.9% in the seven control subjects. The labeled phytanic acid had disapeared in the seven control subjects within forty eight hours, but phytanic acid concentrations in the two subjects with Refsum's disease remained high for many days after. This experiment demonstrated that patients with Refsum's had a block in the degradation of phytanic acid and that even low intake of phytol and phytanic acid could cause an accumulation of phytanic acid in affected patients. Steinberg also showed that phytanic acid undergoes oxidative decarboxylation, producing carbon dioxide and pristanic acid which can readily be β-oxidized as any 2-methyl branched-chain fatty acid.

Phytanic acid is a isoprenoid lipid derived from the phytol side chain of chlorophyll by bacterial degredation. Phytanic acid is derived from phytol through the process of oxidation. The bacterial degredation of chlorophyll to phytol is not preformed in humans, but is preformed in bacteria of herbivores of pelagic fish that then absorb the phytol. In October of 1964 a patient was studied who was on a standard hospital diet. The patient was given Mevalonic acid-2-14C intravenously after an overnight fast and blood samples were drawn from another vein at timed intervals. The hopes of this experiment was to demonstrate endogenous biosynthesis of phytanic acid from mevalonic acid in humans, but their was virtually no incorporation of carbon fourteen into plasma phytanic acid. This disproved the hypothesis of endogenous biosynthesis in humans. Endogenous biosynthesis of phytanic acid has never been documented in humans and it is generally agreed that phytanic acid is ingested from the adipose tissue and muscle of ruminants, invertebrates and pelagic fish. The average human daily dietary intake of phytanic acid in western societies is fifty to one hundred milligrams of which approximately fifty percent is absorbed and metabolized. Phytanic acid is transported in the plasma allied to very low density lipoprotein with its elimination from tissue stores occurring by mechanisms associated with reverse cholesterol transport. Shortly after Daniel Steinberg's study and the study of endogenous biosynthesis of phytanic acid it became apparent that the accumulation of phytanic acid in patients with Refsum's disease could be controlled with dietary modifications. Dietary treatment of Refsum's disease was started in Norway in 1966 by Lorentz Eldjarn who was the head of the Central Laboratory and Institute for Clinical Biochemistry at the Oslo University Hospital, Rikshospitalet. Preliminary results show that at least plasma phytanic acid concentrations could be depleted. Two patients were observed for over a period of a year during which their diet was modified to exclude foods containing phytanic acid or phytol. After several months there was little change, but the plasma phytanic acid concentrations began to fall and went down to levels about one fourth of the initial values.

Through these initial studies as well as subsequent studies of patients with Refsum's disease much has been revealed about the genetic disorder. Refsum's disease is an autosomal recessive disorder. This means that the disorder can only occur if both parents are carriers of the recessive trait. If both parents are unaffected carriers there is a twenty five percent chance that a child will suffer from Refsum's disease, a fifty percent chance of being a asymptomatic carrier, and a twenty five percent chance of being completely unaffected by the disease. In 1997 it was discovered that Refsum's disease was genetically heterogeneous. The genes associated with Refsum's disease were identified and mapped to chromosome 10 for most patients and to chromosome 6q22-24 for approximately ten percent of cases. The minority of cases with Refsum's disease have a deficiency of the peroxisome targeting signal type 2(PTS2) receptor protein peroxin 7 encoded by the PEX7 gene. This receptor protein is required for peroxisomal import and recognition of cytoplasmic proteins with peroxisomal targeting signal type 2. Peroxin 7 deficiency leads to mislocalization and cytosolic accumulation of unprocessed phytanoyl-CoA hydroxylase which is a important enzyme in the α-oxidation of phytanic acid to pristanic acid.

For the majority of cases there is a deficiency in the protein product of the gene PAHXwhich is an enzyme that is required for the metabolism of phytanic acid. This is caused by a impaired PAHX. The subcellular organelle that is deficient in the enzyme activity associated with phytanic acid catabolism was unknown. In 1993 the rates of oxidation of phytanic acid in different subcellular organelles were isolated from cultured skin fibroblasts from both control and Refsum's disease patients in order to discover which organelle is linked to phytanic acid catabolism. The rates of oxidation in peroxisomes, mitochondria and endoplasmic reticulum were 37.1+/-2.65, 1.9+/-0.3, and 0.4+/-0.07 respectively. The rates of oxidation in mitochondria and endoplasmic reticulum were similar in both control and Refsum's disease fibroblasts, but oxidation of phytanic acid in Refsum's disease peroxisomes was non-existent. Refsum's disease peroxisomes had normal rates of oxidation of palmitic acid and lignoceric acid which means that the peroxisomes studied were metabolically active. This experiment discovered that the accumulation of phytanic acid in patients with Refsum's disease is due to a deficient activity of the peroxisomal alpha oxidation enzyme system. In 1995 Mihalika discovered a peroxisomal enzyme phytanoyl-CoA hydroxylase that is deficient in patients with Refsum's disease. Because of the presence of a methyl group on the third carbon phytanic acid cannot undergo β-oxidation. Phytanic acid therefore has to undergo α-oxidation where the terminal carboxyl group is removed to yield pristanic acid. In a normal human phytanic acid reacts with acyl-CoA synthetase, ATP, and CoASH to form phytanoyl-CoA, ADP and PPi. This process occurs on the peroxisomal membrane. Phytanoyl-CoA then reacts with oxygen, 2-oxogluterate, and phytanoyl-CoA hydroxylase with Fe(II) to form carbon dioxide, succinate and 2-hydroxyphytanoyl-CoA. This step in patients with Refsum's disease does not occur due to deficient phytanoyl-CoA hydroxylase. 2-hydroxyphytanoyl-CoA then reacts with 2-hydroxyphytanoyl-CoA lyase to form formyl-CoA and pristanal. Pristanal is then oxidized by aldehyde dehydrogenase and NADP to pristanic acid. These processes occur within the peroxisome. The deficiency of the enzyme phytanoyl-CoA hydroxylase causes phytanic acid to accumulate in the body and affect myelinization. Phytanic acid is readily incorporated into phospholipids. The incorporation of phytanic acid into lipids of the myelin sheath leads to a less stable myelin structure. The methyl branching of phytanic acid disrupts the packing of the hydrocarbon tails in the bimolecular lipid leaflet due to steric hindrance, and in this way destabilizes the myelin. If phytanic acid levels reach the critical point the dissolution of the myelin sheath occurs. The phytanic acid concentrations are generally higher in the peripheral nerve myelin so it starts and is more severe in the peripheral nerve system then in the central nerve system. This destabilizing affect could be the cause of round myelin bodies observed by Cammermeyer in Schwann cells and is the cause of slowed conductivity in nerve cells.

Refsum's disease is one of the few inherited metabolic diseases with an exogeneous cause. This factor makes treatment possible through dietary modifications, but weight management is crucial for treatment of Refsum's disease. A large quantity of phytanic acid is present in body fat stores and during increased mobilization of body fat phytanic acid is liberated from body fat stores. During this mobilization plasma levels of phytanic acid are increased even though intake of phytanic acid is low. This means that during periods of serious weight loss worsening of conditions and even death may occur. Strict diet that helps maintain body weight and reduce phytanic acid intake can be benificial to Refsum's disease patients. Reduction of dietary Phytanic acid is successful in ameliorating some symptoms, but can not fully reverse the progression of the disease. Newer more efficacious therapies such as extracorporeal therapy can be more effective. Phytanic acid can be eliminated by plasmapheresis or apheresis which reduce plasma phytanic acid levels by fifty to seventy percent and can even eliminate phytanic acid form fat stores in some cases. Treatment however has uncertain effects on the progression of retinitis pigmentosa although treatment does seem to stabilize these signs. Due to the fact that Refsum's disease is the only exogenously caused retinitis pigmentosa and a simple cheap biomarker exists it could be possible for screening all patients with retinitis pigmentosa for Refsum's disease. The rarity of the condition however does not make this particular process reasonable. There has been less than three hundred cases of Refsum's disease reported since its discovery in 1946. Testing every individual who is suffering from night blindness for Refsum's disease before any other symptoms are present would be helpful, but the probability of a patient suffering from Refsum's disease would be so small that the test would practically be unwarranted. As w-oxidation is capable of large increases in activity and is most mediated through CYP enzymes, it forms a good candidate for therapeutic interventions to induce enzyme activity and reduce phytanic acid levels in Refsum's disease, but at the moment no drug therapy trials of compounds capable of inducincing w-oxidation have been conducted in humans.

It took the scientific community nearly forty years to fully understand the causes and affects of Refsum's disease, the study of Refsum's disease has lead to many discoveries in biochemistry. The pathways and metabolism of phytanic acid as well as the source of phytanic acid in humans were discovered due to the study of this rare disease. The process of peroxisomes has also been illuminated by the study of Refsum's disease.