Summary of the 2012 PXE Research Meeting

June 28, 2013
By Nick Allen, Research Intern, PXE International, and
Abbie Moore, Research Director, PXE International

Uitto J, Váradi A, Bercovitch L, Terry PF, Terry SF. Pseudoxanthoma elasticum: Progress in research toward treatment: Summary of the 2012 PXE International Research MeetingJ Invest Dermatol. 2013;133(6):1444-9.
 


On September 24-25, 2012, PXE International hosted the 5th international PXE Research Meeting in Bethesda, MD. Leading researchers in the study of pseudoxanthoma elasticum (PXE) gathered to present state-of-the-art, up-to-date information on research and potential treatments for PXE. The main findings are summarized below.
 


Novel Genes, Unusual Phenotypes


PXE is caused by mutations in the ABCC6 gene. Due to advancements made in DNA sequencing, it is possible to discover the two mutations in ABCC6 present in about 90% of the individuals affected by PXE. These advances in sequencing are also helpful in examining new mutations in ABCC6 as well as discovering other genes that might play a role in either PXE or related conditions. PXE International developed and maintains a public database to record all known ABCC6 mutations, housed at the National Center for Biotechnology Information.

Recent biomedical research has also discovered a number of mutations in genes other than ABCC6 that can cause mineralization in the body (Li et al., 2012a). Studying mutations in these genes in conjunction with ABCC6 may lead to a better understanding of how specific mutations in ABCC6 can affect mineralization of different systems in the body. One such example is the ENPP1 gene, which can cause generalized arterial calcification of infancy (GACI). If mutations are present, GACI produces vascular mineralization, similar to what some individuals with PXE experience. However, the vascular mineralization due to GACI occurs prenatally or at birth, while the vascular mineralization due to PXE usually occurs later in life. GACI is a completely separate gene, but the body has a limited number of phenotypes to express genetic alterations.

 


Model Systems


Usually a model system is created using non-human organisms, such as a mouse, that can be studied to learn more about diseases when it is not possible to perform the same experiments on humans. For the study of PXE, strains of knockout (KO) mice have been developed that mimic symptoms that are commonly seen in humans with PXE. These PXE KO mice have been used extensively in research and have greatly contributed to our understanding of PXE.

Recently, several strains of inbred mice were discovered that better mimic the varying degrees of mineralization that humans with PXE exhibit (Li et al. 2012b; Sundberg et al., 2011). While PXE KO mice are experimentally altered to mimic PXE in humans, the inbred mice strains naturally show varying degrees of PXE-like mineralization. Because of this, they are a good model system for examining potential modifier genes. Modifier genes are genes that alter the effect that another gene produces. In the case of PXE, modifier genes may help explain the variation and degrees of symptoms that individuals with PXE experience.

Other new model systems, including insect cell membranes and zebra fish, have been developed to study specific mutations in the ABCC6 gene that cause PXE (Le Saux et al., 2011). Some researchers think that a missing protein in blood causes the characteristic mineralization found in PXE. These mutations are being studied as potential candidates for correction using pharmacological compounds. 

 


Pathomechanistic Pathways


The steps to a pathology, or disease, are called the pathomechanism. Pathomechanisms are studied in PXE research to understand how PXE and its symptoms develop.

Recent studies have examined the role that vitamin K has on the severity of PXE-related symptoms (Borst et al., 2008). It was previously thought that a vitamin K deficiency might contribute to the mineralization that people with PXE experience. Researchers tested this by administering vitamin K to PXE KO mice (Brampton et al.,2011; Gorgels et al., 2011). They found that an increase in vitamin K did not reduce mineralization, suggesting that vitamin K is not a viable treatment for PXE. Find more information on the vitamin K study.

The effect of warfarin, a commonly prescribed blood thinner, on PXE KO mice was also examined (Palaniswamy et al., 2011). Warfarin is known to cause an increase of mineralization in arteries and heart valves. Because of this, it was hypothesized that PXE KO mice on a diet supplemented with warfarin would show more mineralization than PXE KO mice on a normal mouse diet. Results of the study supported this hypothesis. This may mean that individuals with PXE who are also taking warfarin may experience more mineralization than those not taking an anti-coagulant medication. Find more detailed information on the warfarin study.

A finding published just before the 2012 PXE Research Meeting suggested a new pathomechanism for the action of the ABCC6 gene (Martin et al., 2012). Previous research reported that ABCC6 is found mainly in the plasma membrane, the outer lining, of liver cells. The new finding suggested that the ABCC6 gene may be mainly active inside liver cells, rather than in the plasma membrane of those cells. Specifically the authors suggested that ABCC6 gene activity is located in the mitochondria associated membranes (MAMs) of liver cells. MAMs provide most of the cell’s energy, and have a role in the transfer of calcium and other minerals throughout the cell. If the findings on MAMs are found to be credible, it would change the way in which researchers study PXE and how new treatments for PXE are developed. However, a study completed after the publication of this article did not support the hypothesis that the ABCC6 gene is mainly active in MAMs (Pomozi et al., 2013). Instead, results clearly supported previous research that ABCC6 is found in the plasma membrane of the liver. 

 


Phenotypic Modulation


PXE symptoms are known to be variable within the PXE population and even within families. This suggests that environmental factors may affect how PXE progresses. Phenotypic modulation refers to how PXE symptoms are expressed, such as the age of onset, degree of tissue mineralization, and the severity of organs affected. Phenotypic modulation depends on genetic factors, but it also depends on the environment in which the person with PXE lives. Environmental factors that can affect the PXE phenotype include diet, exercise and whether the affected individual smokes.

Studies show that there is no direct correlation between symptoms someone with PXE experiences (phenotype) and specific mutations that cause PXE (genotype). However, there may be a relationship between single-nucleotide polymorphisms (SNPs) and PXE symptoms. Single nucleotide polymorphisms are mistakes in our DNA that affect a single building block. While causing only a small physical change in the DNA, SNPs can have big consequences when affected pieces of DNA are used to create proteins.

Several studies have examined German individuals affected by PXE for SNPs in genes other than ABCC6 that are thought to affect PXE or cause similar symptoms (Zarbock et al., 2009; Zarbock et al., 2010). These studies found that several changes to the DNA sequence are associated with PXE symptoms, severity of symptoms, and the course of the disease. One particular SNP in the MGP gene was even found to potentially be a protective factor in PXE (Hendig et al., 2008). This means that if individuals with PXE have this particular SNP in their MGP gene, they may not experience all symptoms associated with PXE.

 


Gender Differences in PXE Manifestations


In 2011, PXE International conducted an in-depth survey of over 500 affected individuals to better understand PXE, including the gender differences that are commonly reported. Females were disproportionately represented in the study with a total of 393 females and 120 males. The study also revealed several gender differences with regard to PXE symptoms: 1) females are more likely to have skin abnormalities, 2) males are more likely to have severe vision loss, and 3) males are more prone to heart disease associated with PXE. There was no significant difference in diagnosis of peripheral vascular disease between males and females.

 


Toward Treatment


While there are no known treatments for PXE, there has been progress in treating the retinal bleeding associated with PXE. Avastin®, Lucentis®, and Eyelea® are three drugs commonly administered by ocular injection that continue to be effective in preventing the choroidal neovascularization that causes vision loss in PXE.

In addition, a two-year clinical trial testing the effects of magnesium supplementation on mineralization in PXE started in January 2013 with 44 participants. The trial is sponsored by Dr. Mark Lebwohl at the Mount Sinai School of Medicine, and PXE International is a partner in the trial’s recruitment and research. Based on studies in which the magnesium in the diet of PXE KO mice was either increased or restricted, researchers hope to discover whether magnesium supplements might halt or reverse the progression of mineralization in individuals with PXE. Find more detailed information on the magnesium supplement clinical trial.  Find more information on the mouse studies with magnesium.

Researchers are also still in search of a biomarker for PXE. A biomarker is a physical trait or symptom that can be measured in order to track the progression of a disease over time. While physicians can utilize skin biopsies and clinical observations to help track PXE symptoms in humans, such observations can be slower to occur and less indicative of the effects of a treatment. Recent research has shown that the carotid intima-media thickness (CIMT) may be a reliable biomarker for PXE (Kupetsky-Rincon et al., 2012). Carotid intima-media thickness is the measure of the thickness of the inner layers of the blood vessels that carry blood away from the heart. In studies with PXE KO mice, CIMT was found to be thicker in PXE KO mice and thinner in normal mice. Further, CIMT was found to be thicker in PXE KO mice that consumed less magnesium and thinner in PXE KO mice that consumed a diet with increased magnesium. While more research is needed on the topic, researchers believe CIMT holds promise to be a predictive biomarker in humans with PXE.

Research into molecular correction of nonsense mutations has also begun (Y Zhou et al., Unpublished). Nonsense mutations in ABCC6 occur when there is a change in one’s DNA sequence such that production of the protein responsible for stopping mineralization in the body is prematurely ended. Therefore, the protein is not produced and mineralization occurs throughout the body. PTC124, a drug that was developed to treat nonsense mutations in other diseases such as cystic fibrosis, was examined for its effectiveness in correcting nonsense mutations in PXE. Results showed that PTC124 is somewhat effective, but is not efficient. More research is needed to discover better treatments for nonsense mutations.

Recent studies also explored stem cells as a therapy for PXE (Jiang et al., 2012). Researchers injected stem cells into PXE KO mice and results showed that stem cells were able to properly develop into liver cells and express the ABCC6 gene. The ability of these new cells to express the gene, even when the original cells of its host do not have functional copies of the ABCC6 gene, shows a potential for these stem cells as a therapy for PXE. Find more detailed information on the stem cell study.

 


Conclusions


The research on PXE and treatments has come a long way since the last international PXE Research Meeting in 2010. There are, however, many questions still to be addressed about PXE: What is the substrate of ABCC6? Is it possible to develop treatments specific to certain mutations to make the protein responsible for PXE functional? Is stem cell therapy a viable option for human treatment of PXE? What is the biomarker for PXE? We hope to be able to answer all of these questions in the coming years and the work currently being done shows a promising outlook for this goal.

Many of the experiments proposed at this meeting and since are really worthwhile. We invite you to support PXE research by making a donation or using creative ideas to help fundraise in your community.

 


 References


Borst P, van de Wetering K, Schlingemann R. Does the absence of Abcc6 (multidrug resistance protein 6) in patients with pseudoxanthoma elasticum prevent the liver from providing sufficient vitamin K to the periphery? Cell Cycle. 2008;7(11):1575-9.

Brampton C, Yamaguchi Y, Vanakker O, et al. Vitamin K does not prevent soft tissue mineralization in a mouse model of pseudoxanthoma elasticum. Cell Cycle. 2011;10(11):1810-20.

Gorgels TG, Hu X, Scheffer GL, et al. Disruption of Abcc6 in the mouse: Novel insight in the pathogenesis of pseudoxanthoma elasticum. Hum Mol Genet. 2005;14(13):1763-73.

Hendig D, Zarbock, R, Szliska C, Kleesiek K, Götting C. The local calcification inhibitor matrix Gla protein in pseudoxanthoma elasticum. Clin Biochem. 2008;41(6):407-12.

Jiang Q, Takahagi S, Uitto J. Administration of bone marrow derived mesenchymal stem cells into the liver: Potential to rescue pseudoxanthoma elasticum in a mouse model (Abcc6-/-). J Biomed Biotechnol. 2012;2012:818937.

Kupetsky-Rincon EA, Li Q, Uitto J. Magnesium reduces carotid intima-media thickness in a mouse model of pseudoxanthoma elasticum: a novel treatment biomarker. Clin Transl Sci. 2012;5(3):259-64.

Le Saux O, Fülöp K, Yamaguchi Y, et al. Expression and in vivo rescue of human Abcc6 disease-causing mutants in mouse liver. PLoS ONE. 2011;6(9):e24738.

Li Q, Berndt A, Guo H, Sundberg JP, Uitto J. A novel animal model for pseudoxanthoma elasticum: The KK/HlJ mouse. Am J Pathol. 2012;181(4):1190-6.

Li Q, Schumacher W, Jablonski D, Siegel D, Uitto J. Cutaneous features of pseudoxanthoma elasticum in a patient with generalized arterial calcification of infancy due to a homozygous missense mutation in the ENPP1 gene. Br J Dermatol. 2012;166(5):1107-11.

Martin LJ, Lau E, Singh H, et al. Abcc6 localizes to the mitochondria-associated membrane. Circ Res. 2012;111(5):516-20.

Palaniswamy C, Sekhri A, Aronow WS, Kalra A, Peterson SJ. Association of warfarin use with valvular and vascular calcification: A review. Clin Cardiol. 2011;34(2):74-81.

Pomozi V, Le Saux O, Brampton C, et al. ABCC6 Is a basolateral plasma membrane protein. Circ Res. 2013;112(11):e148-51.

Sundberg JP, Berndt A, Sundberg BA, et al. The mouse as a model for understanding chronic diseases of aging: The histopathologic basis of aging in inbred mice. Pathobiol Aging Age Relat Dis. 2011;1.

Uitto J, Váradi A, Bercovitch L, Terry PF, Terry SF. Pseudoxanthoma elasticum: Progress in research toward treatment: Summary of the 2012 PXE International Research Meeting. J Invest Dermatol. 2013;133(6):1444-9.

Zarbock R, Hendig D, Szliska C, Kleesiek K, Götting C. Vascular endothelial growth factor gene polymorphisms as prognostic markers for ocular manifestations in pseudoxanthoma elasticum. Hum Mol Genet. 2009;18(17):3344-51.

Zarbock R, Hendig D, Szliska C, Kleesiek K, Götting C. Analysis of MMP2 promoter polymorphisms in patients with pseudoxanthoma elasticum. Clin Chim Acta. 2010;411(19-20):1487-90.