Summary of the 2010 PXE International Research Meeting


July 28, 2011
By Lisa Ratanaprasatporn, Intern, PXE International


In November 2010, prominent PXE researchers from around the world gathered together in Bethesda, Maryland to attend a two-day research conference hosted by PXE International. At this meeting, the latest research in diagnosis and treatment of pseudoxanthoma elasticum (PXE) was presented and discussed. In March 2011, Dr. Jouni Uitto, Dr. Lionel Bercovitch, Sharon Terry, and Patrick Terry published an article called “Pseudoxanthoma Elasticum: Progress in Diagnostics and Research Towards Treatment, Summary of the 2010 PXE International Research Meeting” in the American Journal of Medical Genetics. The paper documents the very exciting progress in PXE research presented at the conference. The main findings and ongoing research efforts in the understanding of pseudoxanthoma elasticum and activities toward the development of a treatment for PXE are summarized below.
 


Diagnosing Pseudoxanthoma Elasticum (PXE)


Pseudoxanthoma elasticum is an inherited disorder that causes mineralization of the elastic tissue in several systems of the body, particularly the eye, skin, and cardiovascular system. It is estimated that one in 50,000 people has PXE and one in 150-300 people is a carrier. In 2000, it was discovered that mutations in the ABCC6 gene cause PXE. Pseudoxanthoma elasticum is an autosomal recessive disorder, which means that mutations in both copies of the ABCC6 gene in DNA are needed to have PXE. If a person has a mutation in only one copy of the ABCC6 gene, he or she is considered a carrier of PXE. A carrier is not likely to experience PXE symptoms, but has a 50% chance of passing on one copy of the mutation to his or her offspring. Genetic testing for PXE is now commercially available in the US, but it has limitations as it is not able to detect two mutations, required for a confirmed genetic diagnosis, in all affected individuals. Although the testing can be done to diagnose affected individuals before symptoms develop, there are ethical concerns and unintended consequences associated with pre-symptomatic testing. Testing should ideally be accompanied by genetic counseling and an informed consent process.

Diagnosing pseudoxanthoma elasticum can be a challenge for practitioners. Signs of PXE are seldom present at birth, and skin findings usually are not recognized until a patient is in his or her 20’s or 30’s. Thus, an accurate diagnosis is often not made until after several years of delay, sometimes when serious eye or vascular complications develop. Even though PXE appears to be a disease with complete penetrance, there are significant differences in the severity and age of onset of symptoms, even within families. For example, some patients predominantly have skin manifestations with little cardiovascular involvement, while others have severe clinical cardiovascular or severe eye involvement with mild skin findings. Reasons for these differences are not completely understood, but there are recent suggestions that certain mutations in the ABCC6 gene may be statistically associated with involvement of specific target organs. These observations suggest that genetic background, epigenetics, diet, and lifestyle are likely to influence the severity of the symptoms and the age of onset of pseudoxanthoma elasticum.
 


Animal Models


Considerable progress has been made in understanding the molecular genetics of pseudoxanthoma elasticum, and several promising approaches for treatment are now being explored. To develop treatments that may potentially benefit PXE patients, researchers must first study PXE in animal models. The main model currently being used is the mouse model, in which scientists make a PXE “knockout” (KO) mouse that has both copies of the ABCC6 gene “knocked out” by mutations or deletions. This causes the ABBC6 transport protein to be defective or absent, analogous to what occurs in human PXE. The PXE KO mice mimic PXE in humans and serve as a useful model system to explore the mechanisms of PXE and to test potential treatment possibilities before testing in humans. However, the mouse model has limitations. In addition to being costly and time-consuming to create and maintain, the mice have a long developmental lifespan (relative to other laboratory animals, not humans) and relatively slow onset of the disease. As a result, researchers are looking for a better animal model in which to study pseudoxanthoma elasticum. The zebrafish, a freshwater vertebrate, was examined as an alternate. The zebrafish has been found to not be useful for studying mineralization, but might be useful to study compounds that alter the expression of the ABCC6 gene [1].
 


How Does PXE Develop?


Pseudoxanthoma elasticum is caused by a mutation in the ABCC6 gene, which is expressed primarily in the liver, kidneys, and the intestines. The ABCC6 gene is usually not found in organs affected by the mineralization of PXE, such as the skin, eye, and blood vessels. One theory about how this occurs is called the “metabolic hypothesis”, which states that the faulty ABCC6 gene in the liver causes a cellular pump in the liver to break down. The broken pump then does not transport an anti-mineralization substance out of the liver cells and into the blood stream, where it would normally circulate and prevent calcium and other minerals from depositing in elastic tissue.

The molecules transported by the ABCC6 protein from the liver to circulation are currently unknown. Studies have identified a number of proteins that can act as powerful anti-mineralization factors in the circulation, including fetuin-A and matrix Gla-protein (MGP). Patients with pseudoxanthoma elasticum and PXE KO mice have been shown to have reduced serum fetuin-A and reduced activated MGP levels. These observations suggest that these anti-mineralization molecules might be used as a potential treatment for PXE [2].

Tissue damage by reactive oxygen species (ROS) is also hypothesized to affect the PXE phenotype. ROS are chemically-reactive molecules containing oxygen that can result in significant cell damage. When ROS increases dramatically, a situation known as oxidative stress can result. PXE KO mice have been shown to be under chronic oxidative stress. PXE KO mice also have reduced ability to combat the oxidative stress or reduced total antioxidant capacity. However, animal studies have not shown benefit from antioxidant treatment for PXE. Controlled clinical trials on patients with PXE could provide clearer information on the benefits of antioxidant treatments in pseudoxanthoma elasticum.

It has been suggested that the ABCC6 deficiency in PXE patients may result in reduced concentrations of vitamin K in the blood. While this hypothesis is currently being tested in different model systems of pseudoxanthoma elastsicum, it was first reported at this conference that feeding PXE KO mice high doses of vitamin K had no effect on the level of tissue mineralization. These results suggest that vitamin K supplementation of the diet of PXE patients is probably not effective.
 


Translational Research in PXE: From Bench to Bedside


Several key questions must be answered before PXE research findings can be brought from the laboratory bench to the bedside (“translated”). First, the nature of the molecules transported by ABCC6 must be determined. Once these molecules are identified, more specific ways to counteract the mineralization processes of this disorder can be developed. It should be noted that these theories are based on the assumption that the liver is the primary site of molecular pathology in pseudoxanthoma elasticum. Interestingly, three patients, in whom no ABCC6 mutations could be found, developed PXE after receiving a liver transplant from a deceased donor. However, the role of other tissues that normally express the ABCC6 gene, such as the kidneys, must be studied further.
 


Therapies For Pseudoxanthoma Elasticum


A number of encouraging approaches to counteract manifestations of PXE are being studied. One organ-specific approach to the treatment of PXE currently being used is injection of anti-angiogenic medications, such as Avastin® and Lucentis®, into the eye, which prevents the formation of new blood vessels. Significant improvements in visual acuity can be achieved in patients with PXE with the use of anti-angiogenic agents [3]. In fact, the anti-angiogenic agents appear to be so effective that they have largely replaced previously used methods of treatment such as laser photocoagulation and photodynamic therapy. The National Eye Institute just released the results of a clinical trial to compare Lucentis (ranibizumab) with Avasitin (bevacizumab) for the treatment of wet age related macular degeneration (AMD) and found them to be equally effective. Although the trials did not include patients with PXE, it is likely that the results can be extrapolated to pseudoxanthoma elasticum.

Mineral content of the diet may also play a role in the degree of tissue mineralization in PXE. Early surveys suggested that individuals with a history during adolescence of high intake of dairy products, rich in calcium and phosphate, developed more severe disease later in life. However, this has never been confirmed, and to date there is no strong evidence linking dietary calcium intake with severity of pseudoxanthoma elasticum. Studies in PXE KO mice have shown specifically that the magnesium and NOT the calcium content of the diet can influence the extent of mineralization in peripheral tissues. When magnesium carbonate is added to the mouse diet in amounts that increased the magnesium content by fivefold over the standard diet, mineralization was completely prevented in PXE KO mice. Similarly, an experimental diet with low magnesium accelerated the mineralization process. Calcium or phosphate content of the diet had no effect on mineralization. It is possible that magnesium replaces calcium in calcium phosphate complexes, which ultimately leads to reduced or absent mineralization of tissues. Thus, these findings support the idea that diet modifications, specifically changes in dietary magnesium, might be useful for treatment of patients with PXE. Clinical trials are underway in humans to test this theory.

Phosphate binders are another potential treatment for pseudoxanthoma elastsicum. By binding the phosphate in the digestive tract to form a compound that isn’t absorbed in the blood, phosphate binders help to pass excess phosphate out of the body into the stool. This reduces the amount of phosphate that gets into the blood. Preliminary studies on oral phosphate binders have suggested improvement in the degree of skin manifestations of patients with PXE, but no difference between the placebo and the active drug could be shown. When the ingredients of the placebo were later examined, it was found that both the placebo and the phosphate binder had similar, relatively high amounts of magnesium, suggesting that perhaps both acted as magnesium supplements.

The possibility of mutation-specific, ABCC6 targeted therapies for PXE is under investigation. Strategies being studied include development of drugs that would allow the synthesis of normal, functional proteins usually produced by the ABCC6 gene. At the 2010 PXE Research Conference, it was reported that selected mutations that cause pseudoxanthoma elasticum initially showed a problem with trafficking in the cell leading to misdirection of the proteins produced by the ABCC6 gene [4]. In other words, the proteins are not sent to the right place in the cell. Currently, certain drugs such as sodium 4-phenylbutryate are being investigated in animals to correct the misdirection of ABCC6 proteins.

Other strategies for treatment include gene therapy and upregulation of the ABCC6 gene to increase the synthesis of the molecules it normally produces [5]. Over-expression of anti-mineralization factors like fetuin-A could also be beneficial. Transplantation of stem cells derived from bone marrow or the umbilical cord to correct the ABCC6 deficiency is being considered as well.
 


Conclusions


Pseudoxanthoma elastaicum is an autosomal recessive disorder with complete penetrance. PXE is caused by mutations in both copies of the ABCC6 gene, and the severity of the phenotype and age of onset of the disease are likely affected by genetic background, epigenetics, diet, and lifestyle. Great strides are being made in PXE research, which have led to exciting developments in understanding how to diagnose and treat PXE. Anti-angiogenic agents are now being used to counteract new blood vessel growth in the retina. More clinical studies need to be performed to determine if phosphate binders can potentially benefit PXE patients. Clinical trials are also currently being planned to study whether a diet high in magnesium is effective in counteracting PXE mineralization. However, before effective or specific treatment for pseudoxanthoma elasticum can be developed, further research is needed to answer remaining questions about PXE, including the identification of the molecules transported by ABCC6 and the establishment of the liver as the site of molecular pathology critical for the development of PXE phenotypes.


 References


[1] Li Q, Frank M, Thisse C, Thisse BV, Uitto J. 2011b. Zebrafish: A model system to study heritable skin diseases. J Invest Dermatol. 2011 Mar;131(3):565-71. Epub 2010 Dec 30.

[2] Jiang Q, Dibra F, Lee MD, Oldenburg R, Uitto J. 2010. Over-expression of fetuin-A counteracts ectopic mineralization in a mouse model of pseudoxanthoma elasticum. J Invest dermatol.  2010 May;130(5):1288-96. Epub 2010 Jan 21.

[3] Finger RP, Issa PC,, Schmitz-Valckenberg S, Holz FG, Scholl HN. 2011. Long-term effectiveness of intravitreal bevacizumab for choroidal neovascularization secondary to angiod streaks in pseudoxanthoma elasticum. Retina. 2011 Jul-Aug;31(7):1268-78. [Epub ahead of print; PMID:21386758].

[4] Pomozi V, Fulop K, Yamaguichi Y, Szabo Z, Brampton CN, Arányi T, LeSaux O. 2010. Assisted intracellular trafficking of disease-causing PXE mutation of ABCC6 in vitro and in vivo. Poster Abstract, PXE International 2010 Research Meeting, Bethesda, MD.

[5] Váradi A, Szabo Z, Pomozi V, de Boussac H, Fulop K, Arányi T. 2010. ABCC6 as a target in pseudoxanthoma elasticum. Curr Drug Targets 2011 May;12(5):671-82.