Conclusions and Proposals for Future Work on Variegate Porphyria

Variegate Porphyria – A Short Summary

Variegate porphyria (VP) is a rare, autosomal dominant disease most common in South Africa (Hift, 2012). VP results from a 50% decrease in activity of the enzyme protoporphyrinogen oxidase, which catalyzes the penultimate step of heme biosynthesis. In over 95% of South African cases, this is due to a R59W mutation that impairs the enzyme’s ability to bind FAD, which is required for its activity (Qin, 2011)

Erythropoietic symptoms of VP, such as chronic blistering result from the ability of porphyrins to absorb UV radiation and form reactive oxygen species, resulting in oxidative damage (Sassa, 2006). Reducing exposure to sunlight helps alleviate this aspect of the condition. Hepatic characteristics of VP, including seizures and abdominal pain, result from the ability of the porphyrin precursor aminolevulinate to inhibit GABA release and myelin formation (Meyer, 1998) (Felitsyn, 2008). By eliminating factors that induce heme biosynthesis, often by the activation of ALAS-1 transcription, such as estrogen, fasting, and barbiturates, hepatic attacks can be managed relatively well.

When symptoms do develop, pain from erythropoietic traits can be treated with analgesics while acute attacks can be stopped after IV infusions of hematin or haem arginate and glucose, both of which inhibit heme synthesis. In addition, recent work suggests a diet rich in vitamins E and C may help alleviate the chronic state of oxidative stress in VP patients. Lastly, the success of liver transplants on patients with acute intermittent porphyria and one case with variegate porphyria suggest this may also be a means by which the disease can be handled.

What Next? – The Future of Variegate Porphyria

Despite recent advances on understanding the underlying causes of the erythropoietic symptoms of variegate porphyria, namely the state of oxidative stress in the body of VP patients, more needs to be done to fully understand the pathogenesis of hepatic symptoms. Work by Felitsyn et al. indicate that excess aminolevulinate in the brain is able to inhibit myelin formation by the oxidation of proteins and lipids in nerve cells. However, the exact mechanism by which this occurs is not fully understood. Two possible ways are described in their paper: direct damage to cellular proteins caused by the pro-oxidant structural features of ALA (in which loss of an electron would generate a radical), or increased production of reactive oxygen species from an impaired electron transport chain (ETC) (Felitsyn, 2009). Therefore, a better understand of which of these mechanisms, or possibly another one, is the cause of oxidative damage in neurons by ALA would be invaluable in both understanding VP and developing better forms of treatment.

Work by Ferrer et al. demonstrate that genes involved in regulating ROS formation at the ETC, namely UCP-3, Bcl-1, and SIRT-3, are downregulated in the lymphocytes of VP women (Figure 1) (Ferrer, 2010). This suggests that increased ROS production at the ETC may play a significant role in the pathogenesis of porphyrias like VP. In order to assess if this is also the case for neurons, studies similar to that of Ferrer and coworkers examining the expression of these, and other genes, associated with ROS formation at the ETC could provide a lot of insight into the development of hepatic symptoms. In addition, assessing the ability of ALA to induce oxidative in the absence of the electron transport chain would help confirm if ETC ROS production was the only mechanism, or if ALA radicals play a role as well.

Antioxidants and VP

Figure 1. Expression of UCP-3 (B), Bcl-2¬ (C), an SIRT-3 (D) in healthy women (white) versus porphyria women (black). A # denotes statistical significance. Source: Ferrer, 2010.
Figure 1. Expression of UCP-3 (B), Bcl-2¬ (C), an SIRT-3 (D) in healthy women (white) versus porphyria women (black). A # denotes statistical significance. Source: Ferrer, 2010.

Currently, there is no known cure for variegate porphyria. However, work on acute intermittent porphyria (AIP) shows some promise, particularly in the field of gene therapy. Recently it has been demonstrated that treating murine models of AIP with AMT-020, an adeno-associated virus carrying the wild-type porphobilinogen deaminase (the defective enzyme in this form of porphyria) protected the mice from acute attack symptoms with little to no side effects (Siegesmund, 2010). Based off these results, a phase I clinical trial is expected to begin soon. Based off the potential success for gene therapy in treating AIP, it is likely that doing so for VP and other porphyrias may be beneficial as well. Developing a virus capable of carrying and inserting the wild-type PPOX gene into the host’s genome and testing its effects on murine models of VP could be a first step in assessing this form of treatment.

In recent years, there has been an increasing interesting in using small molecules to activate proteins as opposed to inhibiting them (Bishop, 2009). In 2000, Williams and coworkers demonstrated the ability of imidazole to restore the catalytic activity of a R318A protein-tyrosine kinase C-terminal Src kinase mutant (Williams, 2000). They hypothesize this was successful by the imidazole’s ability mimic the missing positively-charged arginine side chain that is required for optimal catalytic activity. Though the R59W mutation in PPOX is different, as the Trp indole ring is much larger than the methyl group on Ala, the disruption in enzymatic activity is more or less same: the replacement of the positively charged arginine with a nonpolar residue introduces unfavorable electrostatic interactions that disrupt enzymatic activity. Therefore, it begs the question of whether imidazole, or another small molecule such as guanidinium, could be capable of restoring PPOX activity in a similar manner. The first step to doing this would likely involve in vitro studies on the ability of small molecules (Figure 2) to improve the ability of the R59W mutant to bind FAD. Since protoporphyrinogen is hard to isolate and very difficult to make in the lab testing the catalytic activity of PPOX after exposure to small-molecule activators would be a challenge. However, since the issue with the R59W mutant is its inability to bind FAD, a small molecule capable of restoring this interaction would be promising for restoring catalysis as well.

Figure 2. Imidazolium and quanadiunium – two small molecules that may be capable of restoring PPOX activity by mimicking the missing positively-charged arginine side chain.
Figure 2. Imidazolium and quanadiunium – two small molecules that may be capable of restoring PPOX activity by mimicking the missing positively-charged arginine side chain.