The is no defect in alpha-oxidation nor a racemase

The patient, a 5-year-old male, has presented with the
following symptoms. Hypotonia of the legs, linked to problems of the nervous
system or muscles(1). Secondary
enuresis, the onset of urinary incontinence after a child has learnt to control
their bladder(2).Together they
can’t rule out either muscular or nervous system conditions. The lack of
cognitive involvement however does suggest the disorder is a neuropathy. It is
unclear how the abnormal pain in the legs manifests, but could be a result of a
disease of the central nervous system. Many peroxisomal disorders lead to loss
of myelination of nerve cells, resulting in impairment of signal transduction,
therefore it would be reasonable to take blood tests to asses concentrations of
peroxisomal metabolites.

 

Blood EDTA of the patient plasma has been tested for
concentrations of very long chain fatty acids (VLCFA), branched chain fatty
acids and bile acid intermediates in plasma. Branched chain fatty acids,
phytanic acid and pristanic acid, are within the limits of the control. Pristanic
acid is the product of phytanic acid alpha-oxidation(3), that
then goes on to be further oxidised by beta-oxidation, as neither concentrations
are affected it can be deduced that there is no defect in alpha-oxidation nor a
racemase deficiency. Bile acid intermediates, dihydroxycholestnoic acid (DHCA)
and trihydroxycholestnoic (THCA)(4), are both said
to be at 0uM concentrations, either they are not present or at concentration below
the degree of accuracy. In either case this is consistent with the control
values for bile acid intermediates, therefore there is no defect in the bile
acid synthesis pathway. However, there is a marked raise in concentration of
VLCFAs. C26/C22 is raised from a control of 0-0.02uM to 0.06uM, and C26:0 is
increased from 0.45-1.32uM to a value of 3.27uM. This is found in peroxisomal
biogenesis deficiencies (PBD) as well as single enzyme deficiencies in the
beta-oxidation of degradation of VLCFAs(5). Percentage of membrane
plasmalogens, a type of ether lipid, were also taken from erythrocytes. C16:0
dimethylacetale and C18:0 dimethylacetale make up 10% and 17% of the patient erythrocyte
plasma membrane respectively. They are both within the reference values,
indicating no deficiency in the etherphospholipid biosynthesis pathway(6). Taking these
sets of results into account it is unlikely that the patient is suffering with
a peroxisomal biogenesis disorder, however this cannot be completely ruled out without
looking at peroxisome distribution in patient cells compared to a control. As
so far, the only metabolite with a marked change in concentration are the
VLFCAs it is possible that the patient has a disorder in the oxidation pathway to
degrade VLCFAs. A more detailed analysis of enzyme activity/metabolite
production in patient cells is needed to further diagnosis plus peroxisome
distribution analysis.

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Examination of patient fibroblasts has been done in
three parts: dihydroxyacetonephosphate-acyltransferase (DHAPAT) activity,
immunofluorescence imaging of peroxisome membrane protein and peroxisomal
enzyme distribution, and VLCFA production. DHAPAT is required for
etherphospholipid biosynthesis, bound to the peroxisomal membrane, and acts as
a diagnosis marker for peroxisomal biogenesis disorders(7). In the patient
fibroblasts DHAPAT activity is measured at 12.7nmol/(2hr.mg protein), just
higher that the upper limit reference value of 12.3nmol/(2hr.mg protein). This
suggests no deficiency in peroxisomal biogenesis. Fluorescence imaging of
patient using RFP tagged peroxisomal membrane protein 70 kDa (PMP70) and GFP
tagged catalase confirms that there is no peroxisomal biogenesis disorder. This
is shown by small circular green dots from the catalase (a peroxisomal
contained protein) and its co-localisation with PMP70 (found on all peroxisome
membranes). We can now rule out Zellweger syndrome (ZS) and the Zellweger
spectrum disorders, caused by defects in peroxisomal biogenesis factors called
PEX proteins(8). This leaves us
with the possibility of a single enzyme deficiency. Concentration of VLCFAs 22,
24, and 26 carbons long were measured and ratios of 24/22 carbon VLCFA and
26/22 carbon VLCFA calculated. Each value was value was taken twice, and I have
averaged them. Whilst C22:0 and C24:0 are not raised, C26:0 is considerably
higher at 0.90 umol/g protein with a normal upper limit of 0.38 umol/g protein.

The subsequent ratios of C24:0/C22:0 and C26:0/C22:0 are markedly raised at
2.36 and 0.23 respectively. The ratio of C26:0/C22:0 is most significantly
raised as its upper limit is normally 0.07, whereas C24:0/C22:0 is only raised
a little as its upper limit is normally 2.30. These findings are consistent
with X-linked adrenoleukodystrophy (X-ALD)(9), caused by
deficiency in the VLCFA import protein. However, as the activity of the VLCFA
degradation machinery has not been looked at it cannot be confirmed.

 

Beta-oxidation of VLCFAs is
performed by three enzymes in four steps, mutations in genes encoding the first
two have been associated with disorders similar to X-ALD(10)(11). Additional
investigations in patient fibroblasts looking at presence of and activity rate
of the three enzymes has been done. Peroxisomal beta-oxidation rate analysis
shows C16:0 and pristanic acid to be degraded at normal rates, but C26:0 is
only converted at 395 pmol/(hr.mg protein), with minimum normal reaction rate
at 1214 pmol/(hr.mg protein). This confirms the rise in VLCFA concentrations is
due to a defect in their degradation. Immunoblotting for the presence of ACOX1
and thiolase, the first and third enzymes in VLCFA beta-oxidation, shows them
to be present to the same extent as the control cells. ACOX1 activity is normal
at 92 pmol/(min.mg protein), control is 35-143 pmol/(min.mg protein). The
second enzyme performs two functions, hydratase and dehydrogenase, and is thus
called D-bifunctional protein (DBP). Both activities of DBP are found to be
preforming at normal rate, hydratase at 375 pmol/(min.mg protein) (control is
115-600 pmol/(min.mg protein)), and dehydrogenase at 188 pmol/(min.mg protein)
(control is 25-300 pmol/(min.mg protein)). These results show that there is no
defect in the beta-oxidation of VLCFAs, however they are not being transported
into the peroxisomes for degradation. Imaging to reveal the distribution of the
protein responsible for VLCFA transport into the peroxisome should reveal if
there is a deficiency.

 

ATP-binding cassette-transporter
(ABCD1) also known as adrenoleukodystrophy protein (ALDP) is required for
import of VLCFAs and their acyl-CoA counterparts into peroxisomes for
degradation(9). Immunofluorescence
imaging of ALDP in patient fibroblasts shows most cells are unstained thus
lacking ALDP. Some cells however are showing some normal staining suggesting there
isn’t a complete lack of protein, the reason should be revealed by sequence
analysis of ABCD1 gene.

 

ABCD1 is located on the X chromosome with 11 exons, as
the patient is male any mutations will be hemizygous. Genetic investigations
reveal a single nucleotide polymorphism mutation within an intronic sequence 10
positions upstream of the next exon whereby guanine has been changed to
adenine. This has the
potential to cause a splicing defect, the resulting protein may then be
targeted for proteasome degradation.

 

From the data, we can see that
there is an accumulation of VLCFAs in plasma and fibroblasts, but no deficiency
in the beta-oxidation pathway, the diagnosis is confirmed by visualisation of
the protein deficiency and final genetic testing showing a point mutation
causing a splicing defect in the ABCD1 gene. From the evidence above, the
patient can be confidently diagnosed with X-linked adrenoleukodystrophy, the
most common of all peroxisomal disorders. X-ALD is characterised by the lack of
VLCFA catabolism due to lack of import into peroxisomes, resulting in their
accumulation in complex lipids causing aggressive demyelination and damage to
the adrenal glands. In particular, the clinical phenotypes match with the most
severe form, cerebral X-linked adrenoleukodystrophy.

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