In our cells, specialized compartments exist that fulfil specific functions. For example, the cell nucleus contains the genetic information in its chromosomal DNA. The so-called endoplasmic reticulum is a structure in the cell that is heavily involved in producing proteins using the information provided by the nucleus. Some of these proteins are called enzymes, implicating that they take care of a particular chemical conversion. Cells can produce numerous enzymes, each involved in one specific step in cellular building and degradation events. For example, specialized enzymes cleave of a particular small fragment from a larger molecule. These degradation enzymes are of vital importance. In cells, most large molecular constituents, like lipids, are continuously produced. In order to remain a balance, continuous degradation of macromolecules by the action of specific enzymes is required. The cell contains specialized compartments for degradation, named lysosomes. In these structures degradation of macromolecules takes safely place. Lysosomal degradation products are often re-used for building purposes, an elegant form of re-cycling at the cellular level. In man, several inherited disorders occur due to impaired re-cycling of macromolecules in lysosomes. In most cases this is caused by defects in one lysosomal degradation enzyme as a consequence of faulty genetic information for its production. Impaired degradation of a natural macromolecule in lysosomes causes its accumulation, which ultimately results in symptoms.

Fabry disease is an X-linked inherited disorder due to abnormalities in the gene encoding the degradation enzyme alpha-galactosidase A. The main function of this enzyme is to degrade a particular lipid, named globotriaosylceramide (Gb-3) or ceramidetrihexoside (CTH) in lysosomes. Since the gene for alpha-galactosidase A is located on the X-chromosome, cells in males only possess just a single copy of this gene. In cells of females, two copies of the X-chromosome are present, and consequently two copies of alpha-galactosidase A gene. However, one of the X-chromosomes in cells of females is inactivated early in life, and its genetic information is no longer available. The X-chromosome inactivation is a complex and more or less random process. Female carriers for Fabry disease (so-called heterozygotes) have cells with two X-chromosomes, one with the correct gene and one with an abnormal gene for alpha-galactosidase A. Depending on which X-chromosome is inactivated, some cells in a heterozygote will be able to produce normal enzyme and others will be not. Generally one observes about half the normal amount of alpha-galactosidase A in plasma and tissues of Fabry heterozygotes. In the case of males with Fabry disease, so-called hemizygotes, only a single copy of the alpha-galactosidase A gene is present, and all cells lack the possibility to produce normal alpha-galactosidase A. As a consequence of this, male Fabry hemizygotes encounter major problems with degrading the lipid Gb-3. The accumulation of the lipid Gb-3 in several types of cells is thought to result in the clinical complications characteristic for Fabry disease. Puzzling for clinicians and researchers is the common occurrence of clinical manifestations in female heterozygotes. In their tissues about half the cells are normally producing alpha-galactosidase A. Usually this is sufficient to prevent any disease manifestation in comparable X-linked disorders such as Hunter disease.

Fabry disease offers another puzzle to clinicians and researchers. The amount of accumulated lipid CTH in individuals seems not to correlate strictly with severity of disease manifestation. For example, in a mouse model of Fabry disease, male animals show massive Gb-3 concentrations in plasma and tissues already immediately after birth without a large further increase with age. Disease manifestations occur much later in life in the animals than lipid storage. The same seems to hold for humans. Young Fabry males show high concentrations of Gb-3 in plasma and urine, clearly proceeding symptoms. Moreover, the extent of Gb-3 elevation in plasma or urine is poorly predicting disease severity. A poor relation between Gb-3 accumulation and disease severity is also noted in female heterozygotes. For example, plasma Gb-3 in female heterozygotes is quite normal, even in symptomatic individuals. A sound explanation is still lacking for the apparent poor correlation between Gb-3 accumulation and disease symptoms. Poorly understood is also the rapid appearance of Gb-3 storage in male Fabry mice without a major increase with age.

Hans Aerts, professor in Medical Biochemistry at the Academic Medical Center, has been intrigued by these Fabry riddles for some time. The disorder was already studied in the seventies in his department by Professor Joseph Tager. In that period another surprising finding was made. A Fabry patient had succesfully received a kidney transplant and renal function had been satisfactory for years. Upon his sudden death due to an infarct, the kidney was inspected and Gb-3 accumulation was detected. This revealed that despite impressive lipid storage, the kidney still had been able to function sufficiently. The observed Gb-3 accumulation in the kidney was difficult to understand since the donor kidney was shown to be able to produce alpha-galactosidase A. Given all these poorly understood phenomena in Fabry disease, Aerts and co-workers decided to re-think about the mechanism of disease development in this disorder. They hypothesized that maybe not only the lipid Gb-3 is relevant in Fabry disease, but that possibly also some metabolite is secondarily generated from Gb-3 and contributes to the actual disease manifestation. They actively searched for such a metabolite, and the results of their investigations were recently published in the scientific journal Proceedings of the National Academy of Sciences. Reported is the presence of so-called lyso-Gb-3 (globotriaosylsphingosine) in plasma samples of Fabry patients. Lyso-Gb-3 is a soluble compound, in contrast to Gb-3, that can easily move in and out cells. It lacks a hydrophobic (acyl) fragment compared to Gb-3. Lyso-Gb-3 is virtually not detectable in plasma obtained from normal individuals, but relatively high concentrations occur in samples from Fabry males. Also in the case of symptomatic Fabry females, increased levels of lyso-Gb-3 were detected. Compared to Gb-3, the abnormalities in plasma lyso-Gb-3 are far more pronounced in Fabry patients. Measurement of lyso-Gb-3 may therefore offer a useful diagnostic tool. This is presently validated by the analysis of blood samples from a large cohort of Fabry males and females. It is also investigated whether plasma lyso-Gb-3 levels have a better predictive value for disease manifestation and severity. Lyso-Gb-3 may be not an innocent molecule. Related water-soluble lyso-sphingolipids are known to influence properties of cells. It is presently studied whether this is also the case for lyso-Gb-3, at concentrations occurring in Fabry patients. A huge amount of fundamental and clinical research will still be required to obtain solid insight into the role of lyso-Gb-3 in Fabry pathology.

The discovery of lyso-Gb-3 in Fabry patients might also help to finally solve some of the riddles imposed by the disease. Aerts and co-workers noted that part of the lyso-Gb-3 daily leaves the body via secretion in bile. This finding has opened up a new hypothesis, coined ‘the secret road’. Confronted with the inability to degrade Gb-3, Fabry patients might adapt to this by exploiting a secret road to get rid of the excessive lipid. The road implies conversion of Gb-3 to lyso-Gb-3, transport of lyso-Gb-3 to the liver, and subsequent secretion from the body via bile. Such a pathway would explain why Gb-3 seems not to accumulate in patients progressively with age. According to this hypothesis, formation of lyso-Gb-3 would be a metabolic adaptation to the traffic jam caused by alpha-galactosidase A deficiency. An inevitable downside of the pathway might however be that Fabry individuals are long term exposed to lyso-Gb3, causing changes in the vasculature and specific cells. It should be clear that this scenario is still a speculation. Studies are presently conducted in Fabry mice to test the challenging hypothesis. If the excretion of lyso-Gb-3 is indeed a major pathway in Fabry patients, one could try to pharmacologically promote the pathway by increasing biliary secretion or reducing intestinal re-uptake of lyso-Gb-3. Such an improve to support the body to remove lyso-Gb-3 may be useful in addition to enzyme therapy that aims to degrade Gb-3, the primary storage lipid.

Summarizing, the discovery of lyso-Gb-3 has led to exciting follow-up investigations regarding diagnostic and predictive value of this lipid metabolite, as well as studies regarding its impact on disease manifestation.

The publication:

Aerts JM, Groener JE, Kuiper S, Donker-Koopman WE, Strijland A, Ottenhoff R, van Roomen C, Mirzaian M, Wijburg FA, Linthorst GE, Vedder AC, Rombach SM, Cox-Brinkman J, Somerharju P, Boot RG, Hollak CE, Brady RO, Poorthuis BJ. Elevated globotriaosylsphingosine is a hallmark of Fabry disease. Proc Natl Acad Sci U S A. 2008 Feb 26;105(8):2812-7. Epub 2008 Feb 19. PMID: 18287059