Information for clinicians
3 Biochemical and genetic testing
3a Plasma tests
3a(i) Plasma phytanic acid concentration
Definitive diagnosis of adult Refsum's disease (ARD) relies upon measurement of the plasma phytanic acid concentration. No false negative results have been reported. Phytanic acid is usually measured by gas chromatography.
- Most individuals with ARD have markedly elevated plasma phytanic acid concentrations (>200 µmol/L; normal: <10µmol/L)
- In a few individuals plasma phytanic acid concentration may be deceptively low, especially in those with ARD caused by mutations in the PEX7 gene [van den Brink et al 2003a] rather than by mutations in the PHYH gene.
3a(ii) Pipecolic acid concentration
Wierzbicki et al  found that about 20% of individuals with ARD show mildly elevated pipecolic acid concentrations which are possibly associated with certain changes in the PHYH gene.
3a(iii) CSF protein concentration
The normal CSF protein concentatrion in adults ranges from 15 to 50 mg/dl. Values in individuals with ARD are considerably higher. In one Arabic family CSF protein concentration was 101 mg/dl [Fertl et al 2001].
3b Skin fibroblast studies
3b(i) Deficiency of phytanoyl-CoA hydroxylase enzyme activity
Greater than 90% of individuals with ARD have deficiency of phytanoyl-CoA hydroxylase, the enzyme encoded by PHYH that catalyses the conversion of phytanoyl-CoA into 2-hydroxyphytanoyl-CoA, a key step in the breakdown of phytanic acid via alpha-oxidation in peroxisomes. To assess activity of this enzyme, phytanic acid alpha oxidation is first measured in cultured fibroblasts. If alpha oxidation is deficient, the activity of the enzyme phytanoyl-CoA hydroxylase is measured.
3b(ii) Deficiency of the peroxisome targeting signal type 2 (PTS2) receptor
Less than 10% of individuals with ARD have deficiency of the PTS2 receptor encoded by the PEX7 gene. The PTS2 receptor plays a key role in peroxisome biogenesis by catalyzing the transport across the peroxisomal membrane of proteins equipped with a peroxisome targeting signal type 2 (like phytanoyl-CoA hydroxylase). Abnormalities in fibroblasts of such individuals include the presence of an abnormal molecular form of peroxisomal thiolase (44 kDa) and partial deficiency of alkyldihydroxyacetonephosphate synthase [van den Brink et al 2003a].
ARD caused by a deficiency of phytanoyl-CoA hydroxylase enzyme activity and ARD caused by a deficiency of the PTS2 receptor cannot be distinguished by plasma phytanic acid concentration or clinical features.
3c Molecular genetic testing
Mutations in two different genes have been identified in adult Refsum's disease.
- PHYH, the gene that encodes phytanoyl-CoA hydroxylase, is located on chromosome 10p13. About 45% of cases have been linked to 10p13 in some studies [Wierzbicki et al 2000]; we have found that >90% of individuals with ARD have mutations in PHYH. (Waterham, Wanders, unpublished observations).
- PEX7, the gene that encodes the PTS2 receptor, is mutated in less than 10% of individuals with ARD.
3c(ii) Molecular genetic testing for adult Refsum's disease: Clinical method
Full gene sequencing: sequencing of coding exons and flanking intron sequences from genomic DNA for both PAHX/PHYH and PEX7 detects mutations in >95% of individuals (Waterham and Wanders, unpublished observations).
3c(iii) Genotype-phenotype correlations
Manifestations of ARD may vary considerably between affected individuals in a family, i.e. between individuals with identical PHYH mutations. These phenotypic differences are comparable to those between affected individuals from different families. Consequently, no clear phenotype-genotype correlations have been identified as yet. This may be related to the dietary intake of phytanic acid, which is thought to be the toxic compound.
More studies are needed to determine if the phenotype differs between those with ARD due to mutations in PHYH and those with ARD due to mutations in PEX7. Preliminary data suggest that the ARD phenotype due to mutations in PEX7 may be milder [van den Brink et al 2003b].
3c(iv) Testing of relatives at risk
It is appropriate to evaluate the sibs of a proband by measuring plasma phytanic acid concentration before symptoms of ARD occur in order to institute early treatment to reduce plasma phytanic acid concentration.
3d Genetically related disorders
Two individuals with elevation of both plasma phytanic acid concentration and pipecolic acid concentration have been reported [Baumgartner et al 2000,Tranchant et al 1993]. The cause of the elevated plasma pipecolic acid concentrations in both individuals is unknown; it is not clear whether the cause is alterations in the PHYH gene or a separate disorder. Sequencing of the gene encoding L-pipecolate oxidase [IJlst et al 2000] did not reveal mutations in these two individuals (Waterham and Wanders, unpublished observations).
- Baumgartner et al  described a patient with psychomotor retardation and abnormally short metatarsals and metacarpals, but no other signs of ARD. Phytanoyl-CoA hydroxylase enzyme activity was deficient; a homozygous deletion of PHYH causing a frameshift was identified. L-pipecolic acid concentration was elevated in plasma. Microscopy of the liver showed the presence of peroxisomes, although they were reduced in number and increased in size. These abnormal liver peroxisomes lacked catalase. Moreover, in fibroblasts a mosaic pattern of cells with and without peroxisomes was found, in contrast to the peroxisomes in fibroblasts from individuals with ARD which cannot be distinguished from controls by catalase immunofluorescence.
- Tranchant et al  described three members of a family diagnosed with adult Refsum's disease. Two had a significant increase of pipecolic acid concentration in plasma and a fourth individual, a brother, died at 17 years of age from a progressive neurological disorder with unusual clinical and neuropathological abnormalities. Homozygosity mapping [Nadal et al 1995] localized the gene to chromosome 10p. Subsequently these individuals were confirmed to have phytanoyl-CoA hydroxylase deficiency in fibroblasts and disease-causing mutations in PHYH (authors' unpublished observations).
Mutations in PEX7 account for rhizomelic chondrodysplasia punctata type I (RCDP type I), a severe and often lethal disorder characterized by mental retardation, rhizomelic shortening of the upper extremities, dwarfism, and cataract [Braverman et al 2002, Motley et al 2002].
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Page last updated 26 June 2006