History and Metabolic Context

X-linked Adrenoleukodystrophy (X-ALD) (MIM 300100) is a neurodengerative disease that is marked by an accumulation of very long chain fatty acids (VLCFA).  X-ALD has a rate of incidence for both hemizygotes and heterozygotes of 1:16,800 (Bezman et al., 2001).  It presents in several different forms.  The childhood cerebral (CALD) form is the most severe one and will be the focus of this review.  It is characterized by a rapid inflammatory demyelination of the brain in males between the ages of 3-10 years (Moser et al. 2001).  This causes severe neurological damage that presents with behavioral changes, a specific gait, and impaired senses.  Ultimately, this leads to a vegetative and death, usually within three years.

CALD affects approximately 35% of males with X-ALD (Moser et al. 2001).  Adrenomyeloneuropathy (AMN) is the less severe phenotype of X-ALD.  It presents in female heterozygotes and some male hemizygotes.  Many cases of CALD start off as AMN so AMN is known as the “default” phenotype (Moser et al. 2001).  AMN usually presents later in life and the symptoms are less severe.  They include a slow, progressive loss of motor function.  Almost all X-ALD patients have adrenal insufficiency (Addison’s disease) that is marked by a darkened pigmentation of the skin (Moser et al. 2001).  This can be treated with hormone injections and is more of a diagnostic marker than a health threat, especially for CALD.   In most of the literature and for this paper, X-ALD will be used to denote material related to both CALD and AMN.  Research specific to the individual phenotypes will be written as such.

X-ALD is caused by a mutation in the ABCD1 gene, which codes for an ATP-binding cassette transport protein on the peroxisomal membrane.  ABCD1 transports VLCFA into the peroxisome to undergo β-oxidation.  There is not a strong genotype-phenotype correlation in X-ALD as the same mutation can lead to different manifestations among individuals within the same family.  While mild, spontaneous breakdown of myelin occurs in all X-ALD patients, the CALD phenotype is not seen until a rapid, inflammatory demyelination is triggered.  The mechanism behind this is poorly defined and highly debated.


Very Long Chain Fatty Acids Accumulate in X-ALD patients

X-ALD had been described as early as 1910 (Moser et al. 2001), but research did not really begin until 1976 when Igarashi et al. demonstrated that VLCFA accumulated in X-ALD patients.  They determined that the C25 and C26 were the most abundant forms of VLCFA.  Since this had not been described in any other disease, the accumulation of VLCFA was recognized as a unique marker for determining X-ALD (Igarashi et al., 1976).  This observation could not be used to diagnose the patients, however, because this accumulation was only found in the brain and adrenal cortex.  Hugo W. Moser, an important figure in the field of X-ALD, was able to create a plasma assay with which to identify X-ALD patients and carriers (Moser et al., 1981).  Using less that 1 mL of plasma, they were able to determine the concentration of VLCFA, especially C26:0, with gas-liquid chromatography (Figure 1).  It had a low rate of false positives, and there were no false negatives in their study (Moser et al., 1981).  This suggests the overall importance of VLCFA in the neurodegeneration of X-ALD because not only is it a unique marker as Igarashi et al. (1976) demonstrated, but all of the X-ALD patients tested had it.

Figure 1: This demonstrated that the hallmark of ALD was the elevation of VLCFA.  The plasma assay determined from this work is still used today (Moser et al., 1981).
Figure 1: This demonstrated that the hallmark of ALD was the elevation of VLCFA. Levels of VLCFA were shown as a ration to C22, which was not elevated in X-ALD.  The bars represent the different ratio of VLCFA accumulation in no disease (first  on left), disease other than X-ALD (2nd to left), X-ALD heterozygote (2nd to right), and ALD hemizygote (right).(Moser et al., 1981).

More advanced diagnostic tools have been developed, especially for the screening of newborns.  In newborns, measuring VLCFA concentrations in the blood has not proven to be as conclusive as is necessary for early diagnosis of X-ALD.  This means that children with X-ALD may not be diagnosed until they are showing symptoms, which limits or eliminates treatment options (Hubbard et al., 2009).  Lyso-phosphatidylcholine (LPC) is generated from phosphatidylcholine, an important part of cell membranes.  VLCFA, namely C26:0, is incorporated into LPC in the brains of X-ALD patients (Eichler et al., 2008).  C26:0-LPC has become a very important marker for X-ALD because of this.  Hubbard et al. (2009), however, developed an assay that uses LC-MS/MS to measure concentrations of C26:0-LPC in the blood of newborns.  This has been shown to be both highly sensitive and accurate so that nearly all of the newborns who were affected with X-ALD were identified, with few false positives.


ABCD1 Causes VLCFA Accumulation

For many years, the understanding of X-ALD was just that it was caused by VLCFA accumulation and that it is X-linked.  It was accepted an X-linked disorder because the severe form of the disease only affected young males, but what gene was mutated was unclear.  To determine the X-linkage of X-ALD, Migeon et al. (1981) studied a family with glucose-6-phosophate dehydrogenase (G6PD) deficiency that also had a history of X-ALD.  The G6PD gene is found on the X chromosome so the mutations could be tracked along with the X-ALD gene.  It was found that the boys in this family who suffered from CALD always had a G6PD deficiency as well (Figure 2).  This established that X-ALD was X-linked, and that the gene causing X-ALD is located near the G6PD locus on Xq28 (Migeon et al., 1981).

Figure 2: Established X-linkage of X-ALD. Males with X-ALD always had G6PD deficiency. (Migeon et al., 1981)
Figure 2: Established X-linkage of X-ALD. Males with X-ALD always had G6PD deficiency. (Migeon et al., 1981)

This helped uncover the genetic defect that was causing X-ALD.  Using positional cloning, the X-ALD gene was found to code for an ATP-binding cassette transport protein (Mosser et al., 1993).  The reason that VLCFA accumulate then, is that they are not brought into the peroxisome to undergo β-oxidation (Wiesinger et al., 2013).  ABCD1 typically functions as a homodimer.  It is an active transporter that relies on ATP-hydrolysis to transport VLCFA into the peroxisome (Figure 3) (van Roermund et al., 2008).  It shares a homology with the three other peroxisomal membrane transport proteins, ABCD2, ABCD3, and ABCD4.  ABCD2 and ABCD3 are of particular importance because they have been shown to account for residual β-oxidation activity in X-ALD fibroblasts.  This has prompted researchers to look at ways to upregulate ABCD2 and ABCD3 to ameliorate VLCFA accumulation.


Figure 3: ABC Transporter
Figure 3: ABC Transporter. Transports substrates by ATP-hydrolysis.  Click on image to see animation. Obtained from YouTube.

CALD is Caused by VLCFA-Induced Inflammation, Treated by Bone Marrow Transplant

The myelin sheath is a collection of fats and proteins that insulates neurons so that the electrical signals are transmitted efficiently.  A loss of myelin, therefore, causes severe neurological damage because neurons are not able to function properly without myelin.  The demyelination of X-ALD has to do with an accumulation of VLCFA, especially C26:0 (hexacosanoic acid).  Since myelin has a higher percentage of VLCFA than most other cells, it is not surprising that an inability to degrade VLCFA would cause neurological distress (Kihara, 2012).The myelin sheath in the CNS is maintained by oligodendrocytes, and they are shown to have a high expression of ABCD1 (Höftberger et al., 2007).  This suggests that if ABCD1 is defective in X-ALD, then oligodendrocytes would be greatly affected because they would not be able to metabolize VLCFA while repairing the myelin sheath (Hein et al., 2008).

The only effective treatment for CALD has been bone marrow transplants (Shapiro et al., 2000).  This has been shown with transplants from donors and autologous hematopoietic cells that have been corrected using lentiviral vectors (Cartier et al., 2009).  The success of bone marrow transplants in ameliorating CALD is not fully understood.  Most theories, though, revolve around microglia, which are the macrophages of the brain.  Microglia act by phagocytocizing materials and triggering inflammatory responses.  These functions are both implicated in the progression of CALD.