2010: PXE Research Conference

On November 29-30, 2010, PXE International held a sponsored gathering of some of the most prominent PXE scientists in the world. Over the course of this two-day meeting, the large group presented their collective progress to date and their ongoing research efforts in the understanding of pseudoxanthoma elasticum, related scientific areas, and activities toward the development of a treatment. This meeting was funded by PXE International and by an R13 grant from the National Institutes of Health: National Institute of Arthritis Musculoskeletal and Skin Diseases (NIAMS), Office of Rare Diseases Research (ORDR), National Eye Institute (NEI), and National Institute of Child Health Development (NICHD).

Researcher Awards
Presentation Abstracts

Read the summary article published in the American Journal of Medical Genetics: “Pseudoxanthoma Elasticum: Progress in Diagnostics and Research Towards Treatment Summary of the 2010 PXE International Research Meeting.”

Researcher Awards

Ivonne Ronchetti received the 2010 PXE International Research Award for long time dedication to pseudoxanthoma elasticum (PXE). We remember with great awe and fondness the first time we visited her laboratory in Modena and saw dozens of bookshelves filled with binders labled 'PXE'. We have been impressed with her important contributions discerning the pathological conditions of the cell. We regreat that she was unable to join us at this meeting since she was assisting relatives who are ill. Daniela Quaglino accepted in her name.


Jouni Uitto received the 2010 PXE International Research Award for his prolific and comprehensive contributions to the understanding of the condition. These contributions include establishing that pseudoxanthoma elasticum is a metabolic disease, creating and testing animal models and experimenting with potential treatment modalities. We have worked with Dr. Uitto for many years, and appreciate the energy and intelligence he brings to the work. Early on, Dr. Uitto participated in PXE International's large-scale quest to discern the spectrum of mutations in ABCC6 and the establishment of the genetic test resulting from the work. We are grateful for his expertise and commitment.



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Presentation Abstracts

Below you will find brief summaries of the presentations from this meeting, divided into six sections. Click to expand or hide. Speakers who received support, either funding or biological samples, from PXE International are indicated with an asterisk after their names.

Basic Science of ABC Transporters
PXE Basic Science
Human Disease Associated With ABC Transporters
Animal Models
Hypotheses
Clinical Studies

Basic Science of ABC Transporters
Chair: Irwin Arias*, NICHD, NIH

• ABC transporters and their role in human disease
  Michael Dean*, Human Genetics Section, Laboratory of Experimental Immunology/Cancer and Inflammation Program, NCI-Frederick, Frederick, Maryland
  

   

  The ATP-binding cassette (ABC) transporter genes represent the largest family of transporters and these genes are abundant in the genome of all vertebrates. Through analysis of the genome sequence databases we have characterized the full complement of ABC genes from several mammals, other vertebrates and invertebrates. Many ABC genes are involved in human disease, involving nearly all organ systems of the body. In addition ABC genes play an important role in the development of drug resistance in cancer cells. At least 14 ABC genes are involved in Mendelian and complex genetic conditions. Most of these are recessive and many display heterogeneity in terms of presentation of the phenotype(s), age-of-onset, and severity. Strategies to use metabolomics to identify candidate substrates will be presented.
   

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• The genetics of ABCC6 and creation of a NLM rare disease database
  Timothy Hefferon*, NCBI/NLM/NIH
  

   

  Since the discovery in 2000 that mutations in the ABCC6 gene cause pseudoxanthoma elasticum (PXE), over 235 presumed disease-causing mutations have been identified. Also during that time, a theory of pseudominant PXE has been proposed and then refuted, two partial pseudogenes have been identified and investigated, and approximately 200 scientific articles have been published describing the ABCC6 gene and protein and their roles in PXE. To help make sense of this wealth of data, a comprehensive resource summarizing ABCC6 variation is needed. A good mutation database should be comprehensive, user-friendly, searchable, customizable, and free of charge. I will describe the recent development and implementation of a new ABCC6 mutation database, hosted at the National Center for Biotechnology Information at NIH and based on the open-source Leiden Open Variation Database software. Patients and researchers alike want to know whether a specific mutation may lead to a predicted increase (or decrease) in the severity of particular clinical parameter. In addition to serving as a comprehensive catalog of mutations, the new ABCC6 database contains genotype and phenotype information on approximately 200 PXE patients, enabling side-by-side comparison and association of genetic and clinical information. While no genotype-phenotype correlation has yet been found for pseudoxanthoma elasticum, this database will serve as a model for other diseases that may have more marked associations between genetic variation and clinical outcome. Importantly, the ABCC6 LOVD database will also serve as a model of human variome microattribution, an important initiative in the genetics community the goal of which is high-quality, community-based annotation of the human genome. By providing submitters with incentive in the form of immediate public recognition for their contributions, it is hoped that databases such as this one will foster improved annotation of disease gene variation. The new ABCC6 mutation database will not only benefit the PXE community by providing added value to the cumulative store of gene-related and research knowledge, but will also serve as an example of the feasibility and utility of using open-source software to establish a successful research tool as well as a solution to the elusive goal of high-quality disease gene annotation.
  
  *Hefferon TW, NCBI/NLM/NIH, Bethesda, MD, USA, Lamb E, PXE International, Washington, DC, USA, Symmons O, Hungarian Academy of Sciences, Budapest, Hungary, Maglott D, NCBI/NLM/NIH, Bethesda, MD, USA, Nash N, PXE International, Washington, DC, USA, Lee D, NCBI/NLM/NIH, Bethesda, MD, USA, Forman L, NCBI/NLM/NIH, Bethesda, MD, USA, Kattman B, NCBI/NLM/NIH, Bethesda, MD, USA, Shao D, NCBI/NLM/NIH, Bethesda, MD, USA, Beloslyudtsev D, NCBI/NLM/NIH, Bethesda, MD, USA, Fulop K, Hungarian Academy of Sciences, Budapest, Hungary, Aranyi T, Hungarian Academy of Sciences, Budapest, Hungary, Varadi A, Hungarian Academy of Sciences, Budapest, Hungary, Terry S, PXE International, Washington, DC, USA.
   

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• Basic science of ABC transporters
  Irwin Arias*, Unit on Cellular Polarity, Cell Biology and Metabolism Branch, NICHD/NIH, Bethesda, MD
  

   

  My presentation will review what is known and unknown about the cell biology of ABCC6. Examples will be given using other ABC transporters and discoveries made using live cell imaging techniques. The role of transcriptional and translational regulation, protein folding, targeting, membrane insertion, cycling and degradation will be illustrated. Regulation of these processes offers possible therapeutic targets even though the transported ligand for ABCC6 remains elusive. Therapeutic possibilities involving transplantation of cells and/or liver or kidney will be discussed while presenting advances in these fields which may be relevant to ABCC6.
   

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• The ABCC6 protein: modeling and structure-function studies
  András Váradi*, Institute of Enzymology, Hungarian Academy of Sciences
  

   

  Constructing homology models of ABCC6 is now possible as recently high resolution crystal structures of two ABC export pumps were published. There are newly recognized structural elements in the structures, like the long "rigid" extensions of the transmembrane helices, called intracellular loops (ICL). Each half of the ABC proteins has two ICLs interacting with the ABC-domains. The coupling helices at the tips of ICLs contact with their “own” as well as with the “opposite” ABC-domains, hence a special type of domain swapping was recognized. These domain-domain interactions play a crucial role in the intramolecular communication during ATP-fueled transport.
  
  We have constructed two homology models of the human ABCC6 protein: one of the models is based on the Sav1866 bacterial ABC transporter structure [Fülöp et al, 2009, BBRC 379: 706-9] representing a nucleotide-saturated conformational state, while the other one uses the recently published mouse Abcb1 structure as a template and represents the nucleotide-free (apo) conformation [Váradi et al, unpublished]. By performing a statistical analysis we have found a significant clustering of the missense PXE-mutations at the domain-domain interfaces: at the transmission interface that involves four intracellular loops (ICLs) and the two ABC domains as well as at the ABC - ABC interacting surfaces. The observed significant clustering means that the domain contacts are much less permissive to amino acid replacements than the rest of the protein. These results provide a “bridge” between clinical/genetic data and protein structure. We have built homology models of several other clinically important ABC-transporters (ABCB4, ABCB11, ABCC2, ABCC7, ABCC8 and ABCD1); this work revealed similar clustering of missense mutations as we observed in the case of ABCC6.
  
  In spite of the remarkable progress in structural modeling, our recent analyses indicate that rationalizing and predicting the functional impact of missense mutation and non-synonymous SNPs on protein structure and function is difficult, thus underlining the importance of detailed in vitro and in vivo experimental studies. Such studies are performed in collaboration with the Le Saux group (Univ. Hawaii) and will also be presented at the conference.
  
  *A. Váradi, K. Fülöp, L. Barna, O. Symmons, V. Pomozi and T. Arányi
  Institute of Enzymology, Hungarian Academy of Sciences, Budapest, Hungary
   

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• Transcriptional regulation of the ABCC6 gene
  Tamás Arányi*, Institute of Enzymology, Hungarian Academy of Sciences
  

   

  To understand the pathomechanism of pseudoxanthoma elasticum (PXE) and find new treatments are crucial. This is an even more important issue when we consider that other phenotypes are also associated with PXE. Indeed, ABCC6 mutation carriers have an increased risk to develop coronary artery disease; a significant portion of beta-thalassemic patients develop PXE-like phenotype most probably due to the down-regulation of ABCC6 gene expression. PXE-like phenotype can also develop in the ABCC6 mutation carrier when they are carriers of other mutations, e.g. in the GGCX gene. It should be noted that pseudoxanthoma elasticum is also associated with a promoter polymorphism of the gene. Furthermore some mutations do not lead to complete protein function loss, while others have not yet identified mutations, potentially localized in gene expression regulatory regions. All these clinical conditions could be ameliorated by the increase of the expression of the gene. We therefore investigated the transcriptional regulation of the gene.
  
  We used several parallel approaches from the field of molecular biology to understand the tissue-specific regulation of the gene and the potential modulation of the ABCC6 expression by extracellular signals. We performed our studies by comparing ABCC6 expressing (human hepatoma HepG2) and non expressing (HeLa) cell lines. We demonstrated that ABCC6 transcription is downregulated due to oxidative stress and activation of the ERK cascade by extracelular signals. This effect is mediated via HNF4, which orchestrates the tissue-specific expression of the gene according to our findings. HNF4 is a major regulator of gene expression hepatocytes and regulates hepatocyte function according to the metabolic state of the body. We found that ABCC6 is expressed at a considerable level only in tissues where HNF4 is present. We also observed that the gene has a primate specific strong intronic tissue-specific enhancer element, which interacts with the promoter. These data reveal the complex regulatory mechanisms of the ABCC6 gene and open new perspectives towards developing new therapies for PXE and PXE-like phenotypes. Our results open new perspectives for drug development targeting the transcriptional regulation of ABCC6.
  
  *Aranyi, T, Institute of Enzymology, Hungarian Academy of Sciences, Budapest, Hungary, de Boussac, H, Institute of Enzymology, Hungarian Academy of Sciences, Budapest, Hungary, Ratajewski, M, Institute of Medical Biology, Polish Academy of sciences, Lodz, Poland, Sachrajda, I, Institute of Medical Biology, Polish Academy of sciences, Lodz, Poland, Bacquet, C, Institute of Enzymology, Hungarian Academy of Sciences, Budapest, Hungary, Pulaski, L, Institute of Medical Biology, Polish Academy of sciences, Lodz, Poland, Varadi, A, Institute of Enzymology, Hungarian Academy of Sciences, Budapest, Hungary.
   

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PXE Basic Science
Chair: Jouni Uitto*, Jefferson Medical College

• Genotype-phenotype correlations in pseudoxanthoma elasticum: from disease to spectrum of disorders
  Anne De Paepe*, Medical Genetics/Ghent University Hospital
  

   

  The phenotype of pseudoxanthoma elasticum (PXE) is characterized by a significant variability in the severity of the ophthalmological, dermatological and cardiovascular symptoms, not only between families but also between sibs with a similar molecular background. Several attempts have been made to establish relevant correlations between the patients phenotype and genotype, but thusfar no such correlation could be delineated.
  
  The identification of novel phenotypes which are clinically or pathologically or bothwise related to PXE has added to the hypothesis that pseudoxanthoma elasticum is part of a spectrum of diseases. Within this spectrum patients present with similar symptoms while the molecular pathways involved are often different. An overview will be given of PXE-related phenotypes which - although patients presented with PXE symptoms - were proven not to be related to mutations in the ABCC6 gene. It can however be assumed that their individual pathways may be interconnected. As more is known about the molecular background of these disorders, the concept of genotype-phenotype correlation within the PXE spectrum may hold fascinating knowledge on ectopic mineralization disorders and elastic fiber homeostasis.
  
  *De Paepe A.¹, Coucke P.J.¹, Martin L.², Robert L.M.P.³, Turnpenny P.⁴, Vanakker O.M.¹
  1. Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium
  2. Department of Dermatology, Angers University Hospital and UMR CNRS 6214/INSERM 771, Integrated Neurovascular Biology, University of Angers, Angers, France.
  3. Clinical Genetics, Guy’s and St Thomas’ Hospital, London, UK
  4. Clinical Genetics, Royal Devon & Exeter NHS Trust, Exeter, UK
   

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• Pathology of elastic structures: primary or secondary effect?
  Jeffrey Davidson, Department of Pathology, Vanderbilt University School of Medicine and Research Service, VA Tennessee Valley Healthcare System, Nashville, USA
  

   

  Elastic fibers form late in development and during postnatal growth by deposition and crosslinking of soluble tropoelastin on a core of microfibrillar proteins that includes fibrillins, fibulins, and microfibril-associated glycoproteins. Elastic fibers reside in the skin, lungs, and blood vessels for decades. They slowly degrade due to biochemical, mechanical and UV-related attack with little or no turnover or regeneration. Diseases involving the assembly of elastic fibers and elastin crosslinking via lysyl oxidases occur perinatally or during early life.
  
  Congenital cutis laxa and Marfan syndrome are the most dramatic examples. During adult life, extensive degradation of elastin occurs in pulmonary emphysema and acquired forms of cutis laxa; moreover, there are a large number of conditions, including pseudoxanthoma elasticum (PXE), where elastocalcinosis accompanies the partial degradation of elastic fibers. In the vasculature, the calcification of medial elastic fibers is attributed to several factors, including imbalanced Ca:P, induction of an osteogenic phenotype in vascular smooth muscle cells, exposure of nucleation sites for mineralization of the fibers, and loss of protective factors such as matrix Gla protein, GGCX and fetuin. Elastocalcinosis is associated with many diseases and syndromes. Vascular calcification is much more localized in PXE, and other calcified lesions are restricted to elastic fibers of the mid-dermis in flexural areas and Bruch’s membrane, both of which are not generally affected in other elastocalcinoses. Given the variable penetrance of pseudoxanthoma elasticum in families, there may be some subtle genetic changes in components of the elastic fiber that predispose elastic tissue to mild, site-specific degradation, and calcification of elastin in vitro is accelerated by metalloproteinases. In addition, PXE serum is reported to cause abnormal assembly of elastic fibers in vitro. Nevertheless, both the lack of developmental onset of PXE and genetic linkage of components of the elastic fiber to the disease are consistent with elastin abnormalities that are secondary to elastic fiber formation. Supported by the National Institute on Aging and the Department of Veterans Affairs.
   

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• Therapeutic approaches to inhibit elastic fiber calcification in PXE: new perspectives derived from treatments of PXE fibroblasts
  Daniela Quaglino*, Dept. Biomedical Sciences- University of Modena and Reggio Emilia - Modena, Italy
  

   

  Background: Dermal fibroblasts isolated from patients with pseudoxanthoma elasticum (PXE) exhibit and maintain in vitro pathologic features, possibly related to calcification, thus representing a useful and informative model to assess the efficacy of compounds for therapeutic perspectives. PXE fibroblasts and plasma are characterized by unbalanced oxidative stress that, in plasma, is related to severity of clinical manifestations and is associated with low vitamin K level. However, the possible role of these alterations in elastic fiber calcification is still not clear.
  
  Methods: Dermal fibroblasts isolated from PXE patients were cultured in vitro and treated with antioxidants (vitamin C, vitamin E, kaempferol, quercetin) and with vitamins K1, K2 and K3. Parameters of oxidative stress and the expression of proteins regulating the gamma-carboxylation system as well as calcium and phosphate homeostasis were evaluated by flow cytometry and Western blot.
  
  Results: Antioxidants efficiently reduced oxidative stress, lowered the expression of tissue non specific alkaline phosphatase (TNAP) without affecting proteins involved in vitamin K recycling and gamma-carboxylase activity (i.e. protein disulfide isomerase and calumenin). Vitamin K3, as a pro-oxidant agent, negatively affected fibroblast viability. At the same doses, vitamin K1 was ineffective on redox balance, whereas vitamin K2 lowered the ROS level in PXE cells. Vitamins K1 and K2 significantly up-regulated gamma-carboxylase activity.
  
  Conclusion: In vitro treatment of PXE fibroblasts with antioxidants and with vitamins K1 and K2 had beneficial effects on parameters that are known to be altered in the disease. Further studies in in-vivo models are necessary to investigate on the efficacy of these compounds to inhibit/delay elastic fiber calcification.
  
  *Quaglino D., Boraldi F., Annovi G., Guerra D., Tiozzo R., Ronchetti I.
  University of Modena and Reggio Emilia, Modena, Italy
   

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Human Disease Associated with ABC Transporters
Chair: Michael Gottesman*, NCI, NIH

• ATP binding cassette (ABC-) transporter proteins are composed of multiple domains that contribute distinctly to their function
  Patrick Thibodeau, Cell Biology and Physiology/University of Pittsburgh School of Medicine
  

   

  Minimally, ABC-transporters are composed of two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs). The TMDs facilitate solute movement across biological membranes while the NBDs bind and hydrolyze ATP to provide energy for these transport processes. In addition to these core domains, multiple ABC-transporters contain additional accessory domains that regulate protein trafficking, protein-protein interactions and transport activities. A growing number of disease-associated mutations have been identified in the ABCC subfamily of human ABC transporters. Disease pathophysiology may arise from biosynthetic, trafficking and/or functional defects, ultimately resulting in alterations to cellular transport activities. Studies of ABCC7 (CFTR) indicate that both local and global structural properties are altered by many disease-associated mutations. Specifically, mutations within the cytosolic nucleotide binding domains result in local alterations in NBD structure and biophysical properties. These alterations subsequently influence the native domain-domain interactions and are propagated globally. Local changes in NBD structure are directly recognized by cellular quality control and critically contribute to the regulation of transporter biosynthesis and trafficking. To better understand how mutations in the ABCC6 NBDs influence biosynthesis and protein function, the NBDs of ABCC6 have been expressed and purified. Highly purified proteins have been subjected to structural and functional analyses. Functional analysis demonstrates ATPase activity in both of the purified ABCC6 NBDs that is disrupted by NBD mutations. Biochemical characterization of the NBDs suggests that local properties/structures may be altered by NBD mutations, resulting in downstream changes in ABCC6 global properties.
  
  Stonebraker, KL, Hergenrother, SD, *Thibodeau, PH, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
   

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• Simple and complex ABCA4 in retinal diseases: from genetic studies to treatment
  Rando Allikmets, Departments of Ophthalmology and Pathology & Cell Biology, Columbia University, New York, NY, USA
  

   

  When the adenosine triphosphate (ATP)-binding cassette (ABC) transporter gene, ABCA4 (originally named ABCR), was cloned and characterized in 1997 as the causal gene for autosomal recessive Stargardt disease (STGD) it seemed as if just another missing link was added to the extensive table of genetic determinants of rare monogenic retinal dystrophies. Now, 13 years later, the ABCA4 gene continues to emerge as the predominant determinant of a wide variety of retinal degeneration phenotypes, such as STGD, cone-rod dystrophy, retinitis pigmentosa, and age-related macular degeneration. ABCA4 has caused exciting and sometimes intense discussions among ophthalmologists and geneticists, resulting in more than 300 publications during this time. In my presentation I will summarize our current knowledge of the role of ABCA4 in retinal disease and review the substantial progress in diagnostic and therapeutic applications for ABCA4-associated disorders which most recently seemed impossible. Although ABCA4 has proven to be a complex and difficult research target, I hope to convince the audience that treatment of all ABCA4-associated disorders, and especially STGD, should be possible in the near future.
   

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Animal Models
Chair: Olivier Le Saux*, University of Hawaii

• Pseudoxanthoma elasticum mouse models
  Qiujie Jiang*, MD. Ph.D., Thomas Jefferson University
  

   

  Pseudoxanthoma elasticum (PXE), characterized by connective tissue mineralization of the skin, eyes, and cardiovascular system, is caused by mutations in the ABCC6 gene, expressed primarily in the liver and the kidneys. Mechanisms leading to ectopic mineralization as a result of these mutations remain unclear.
  
  To elucidate this complex autosomal recessive disease, a transgenic mouse was generated by targeted ablation of the mouse Abcc6 gene. Abcc6 null mice were negative for Abcc6 expression in the liver, and complete necropsies revealed profound mineralization of several tissues, including skin, arterial blood vessels, and retina, while heterozygous animals were indistinguishable from the wild-type mice.
  
  Using this knockout model, in which mineralization of tissue begins about 5 weeks after birth, we were able to clarify several critical features of the disease. So far we have demonstrated that 1) Pseudoxanthoma elasticum has features of a metabolic disease based on information from grafting or parabiotic experiments on these mice; 2) restoration of normal fetuin-A levels in the serum by overexpression of the fetuin-A gene (Ahsg) in the liver may counteract the progressive mineralization in PXE; 3) several factors, including the strain specificity and the genetic background of the mice, as well as the mineral content of their diet, contribute to the severity of PXE.
  
  In summary, Abcc6 null mouse can serve as a model for pseudoxanthoma elasticum, providing a crucial animal system to elucidate PXE pathomechanisms and to test therapeutic interventions. Furthermore, these mice may serve as a general model of mineralization in studies of vascular and ocular degeneration.
  
  *Q. Jiang1, R. Oldenburg¹, F. Dibra¹, S. Otsuru2, M. Endo², Q. Li¹, A. Grand-Pierre¹, J. Uitto1
  1 Thomas Jefferson University, Department of Dermatology and Cutaneous Biology, Jefferson Medical College, and Division of Oncology, 2 Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, 19107, USA
   

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• In vivo expression and maturation of the human ABCC6 and its PXE-mutants in mice liver
  Olivier Le Saux*, John A. Burns School of Medicine, University of Hawaii
  

   

  The main consequences of diseases-causing mutations in ABC transporter are altered activity, protein folding and/or incorrect membrane targeting. ABCC6 mutations cause pseudoxanthoma elasticum (PXE), a disease characterized by calcification in vascular and dermal tissues. The exact function of ABCC6 is still unknown. Therefore, we studied ABCC6 by using naturally occurring PXE mutations to identify residues structurally and functionally relevant. Five mutants with substitutions in the C-terminal NBD and transmission interface were generated: R1138Q, V1298F, R1313W, G1321S and R1339C. Four of these were studied in vitro for transport activity, ATP binding, glycosylation and by trypsin digestion. An N-terminally truncated protein (DeltaC6) was also generated for control purposes. R1138Q, V1298F, R1313W, G1321S were able to bind ATP but only R1138Q and R1314W demonstrated transport activity comparable to the WT protein. R1339C was instable thus preventing functional analysis. Only mutant R1138Q showed limited trypsin digestion patterns different from the WT protein and was partially glycosylated when expressed in MDCKII cells. To improve on these in vitro observations, all 6 mutants were transiently expressed in vivo, in mouse liver via tail vein injections. The human WT ABCC6 was fully integrated in the basolateral membrane of in mouse hepatocytes and co-localized with the endogenous Abcc6. V1298F was the only mutant that showed WT behavior in vivo while all other mutants showed various levels of intracellular accumulation indicating abnormal trafficking. Previous studies have shown that 4-phenylbutyrate (4-PBA) can partially restore cellular trafficking and function of mutanted ABCC7 (ΔF508). Therefore, we tested 2 ABCC6 mutants with residual transport activity (R1138Q and R1314W). We found that 4-PBA improved the membrane localization of R1314W suggesting that the incorrect intracellular trafficking is likely due to protein misfolding and/or ER retention.
  
  *O. Le Saux, K. Fülöp, Y. Yamaguichi, A. Iliás, Z. Szabó, C.N. Brampton, V. Pomozi, K Huszár, T. Arányi, András Váradi
   

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• Elevated dietary magnesium prevents connective tissue mineralization in a mouse model of pseudoxanthoma elasticum (ABCC6(-/-))
  Jennifer LaRusso*, Dermatology/Thomas Jefferson University [biography unavailable]
  

   

  Pseudoxanthoma elasticum (PXE) is an autosomal recessive multisystem disorder characterized by ectopic connective tissue mineralization, with clinical manifestations primarily in the skin, eyes, and cardiovascular system. There is considerable, both intra- and interfamilial, variability in the spectrum of phenotypic presentation. Previous studies have suggested that mineral content of the diet may modify the severity of the clinical phenotype in PXE. In this study, we utilized a targeted mutant mouse (Abcc6(-/-)) as a model system for PXE. We examined the effects of changes in dietary phosphate and magnesium on the mineralization process using calcification of the connective tissue capsule surrounding the vibrissae as an early phenotypic biomarker. Mice placed on custom-designed diets either high or low in phosphate did not show changes in mineralization, which was similar to that noted in Abcc6(-/-) mice on a control diet. However, mice placed on a diet enriched in magnesium (fivefold) showed no evidence of connective tissue mineralization in this mouse model of PXE. The inhibitory capacity of magnesium was confirmed in a cell-based mineralization assay system in vitro. Collectively, our observations suggest that assessment of dietary magnesium in patients with pseudoxanthoma elastsicum may be warranted.
  
  *LaRusso J, Li Q, Jiang Q, Uitto J. Thomas Jefferson University, Philadelphia, Pennsylvania, USA
   

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Hypotheses
Chair: Jouni Uitto*, Jefferson Medical College

• Vitamin K-dependent g-glutamyl carboxylation reactions
  Kathleen Berkner, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio
  

   

  Vitamin K-dependent (VKD) proteins are rendered active when clusters of Glu’s in a domain within these proteins are carboxylated to generate a calcium-binding module. Sixteen VKD proteins have been identified to date, and all of these proteins are modified by a single enzyme, the gamma-glutamyl carboxylase, whose activity depends upon the actions of a vitamin K oxidoreductase and a redox protein that activates the oxidoreductase. Virtually all tissues express at least one VKD protein, and in many cases the expression of multiple VKD proteins has been demonstrated. The functions of the hemostatic VKD proteins are well-established while those of the nonhemostatic VKD proteins are only poorly understood. How the carboxylase activates VKD proteins is also not well-established. Defining the mechanism of carboxylation has been challenging because the carboxylase is a large, integral membrane protein without structural information and because there are no family members to guide structure-function studies. An important direction for understanding carboxylase function has been the analysis of naturally-occurring carboxylase mutations, which give rise either to a severe bleeding defect or to a PXE-like phenotype. The presentation will discuss different hypotheses for how carboxylase mutations can result in two different diseases, and will also describe characterization of two carboxylase mutations whose compound heterozygosity causes the PXE-like phenotype.
   

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• Role of serum fetuin-A: a major inhibitor of systemic calcification in pseudoxanthoma elasticum
  Doris Hendig, Institute for Laboratory and Transfusion Medicine, Heart and Diabetes Center NRW, University Hospital of the Ruhr-University Bochum, Bad Oeynhausen
  

   

  Objective: Pseudoxanthoma elasticum (PXE), a hereditary disease of the connective tissue affecting skin, retina, and cardiovascular system, is histologically characterized by progressive calcification and fragmentation of elastic fibers in the extracellular matrix. We and others have recently found that mutations in the ATP binding cassette transporter subfamily C member 6 (ABCC6) gene, encoding the multidrug resistance-associated protein 6, cause PXE. The implication of ABCC6 in the mineralization process is still unknown. In the present study, we examined the association between fetuin-A - a major inhibitor of systemic calcification - and the PXE phenotype to discover the link between the absence or functional insufficiency of ABCC6 and the calcification of elastic fibers.
  
  Materials and Methods: Fetuin-A was measured by quantitative sandwich enzyme immunoassay in sera from 110 German patients with pseudoxanthoma elasticum, 53 unaffected first-degree family members and 80 healthy control subjects. We determined the distribution of the fetuin-A polymorphisms c.742C>T (p.T248M) and c.766C>G (p.T256S) in these same three groups.
  
  Results: Serum fetuin-A concentrations in male and female PXE patients were lower than in unaffected first-degree relatives and controls (patients, 0.55 ± 0.11 g/L; relatives, 0.70 ± 0.23 g/L; controls, 0.80 ± 0.23 g/L; p <0.0001). Interestingly, the value for serum fetuin-A determined in the group of relatives was intermediate between that of PXE patients and controls.
  
  Moreover, we noticed decreasing fetuin-A levels in association with the number of organs involved, although it did not reach significance. The fetuin-A polymorphism frequencies did not differ between PXE patients, family members and blood donors. However, fetuin-a genotypes affect serum fetuin-a levels.
  
  Discussion and Conclusion: These findings support the hypothesis that a deficiency of ABCC6 leads to an alteration of circulating substrates, e.g. inhibitors of calcification as fetuin A, resulting in a progressive mineralization of elastic fibers in PXE.
  
  *Hendig, D.¹, Szliska, C.², Kleesiek, K. ¹, Götting, C. ^{1
  1}Institut für Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Bad Oeynhausen, Germany
  ²Dermatologische Klinik, Krankenhaus Bethesda, Freudenberg, Germany
   

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• Modifier genes in pseudoxanthoma elasticum
  Olivier Vanakker*, Ghent University Hospital
  

   

  The intra- and interfamilial variability of the pseudoxanthoma elasticum (PXE) phenotype and the difficulties in defining correlations between the ABCC6 mutations and the dermatological, ocular and/or cardiovascular symptoms of patients suggests the possibility of modifier genes playing a role in this complex disease. Some candidate modifiers have already been proposed in literature, which either have an effect on the natural history of the disease or affect a specific subset of symptoms. In the quest for modifying factors, the purpose these factors should serve upon their identification and hence based on which criteria modifiers can be designated relevant or not should be taken into consideration. After all, as the knowledge on the pathophysiology of PXE expands, and the spectrum of PXE-associated diseases grows, so does the number of candidate modifying genes. This abundance of candidates will hold the challenge of selecting those relevant for clinical use. In this presentation, an overview will be given of characteristics modifying genes should probably comply to. Modifiers already available for pseudoxanthoma elasticum will be looked at in terms of these criteria to evaluate their clinical usefulness.
  
  O.M. Vanakker¹, P.J. Coucke¹, J. M. Hosen¹, A. De Paepe^{1
  1}Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium
   

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Clinical Studies
Chair: Lionel Bercovitch, Brown University, Providence, RI

• Treatments for the eye manifestations of PXE
  Emily Chew*, National Eye Institute/National Institutes of Health
  

   

  Ocular manifestations of pseudoxanthoma elasticum (PXE), one of the major morbidities of this condition, consist of angioid streaks, peau d’orange, optic nerve head drusen, retinal pigment and epithelial lesions. The main cause of visual loss is due to choroidal neovascularization secondary to the angioid streaks which are cracks in Bruch’s membrane, tissue that is under the retina. The use of anti-vascular endothelial growth factor (VEGF) has made great improvements in diseases with choroidal neovascularization, mostly age-related macular degeneration (AMD). Drugs such as ranibizumab (Lucentis) and bevacizumab (Avastin) injected intravitreally have been successful in reducing the risk of vision loss and improving the vision by three or more lines in over 40% of patients with neovascular AMD. Although no studies have been conducted in ocular diseases of PXE with anti-VEGF therapy, case reports suggest that these therapies may have beneficial effects in the therapy of choroidal neovascularization associated with pseudoxanthoma elasticum.
  
  The characterization of the different ocular lesions in PXE with the advanced imaging techniques such as optical coherence tomography (OCT) or fundus autofluorescence (FAF) have improved our understanding of the ocular disease. Natural history studies of the ocular disease may also provide important information about the risk factors for the progression of the disease to the advanced stage. Studies of genotype-phenotype correlation may also provide important clues to progression of disease.
   

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• Renagel study
  Mark Lebwohl*, Professor and Chairman, Department of Dermatology, The Mount Sinai School of Medicine
  

   

  Background: Pseudoxanthoma elasticum (PXE) is a systemic connective tissue disorder involving elastic fiber calcification and fragmentation with major clinical manifestations occurring in the cutaneous, ocular, and cardiovascular systems. Normalization of the serum calcium-phosphate product through hemodialysis in a previous patient with perforating periumbilical PXE and elevated serum phosphate resulted in regression of skin lesions.
  
  Objective: We sought to study the effect of pharmacologically limiting the intestinal absorption of phosphate in patients with pseudoxanthoma elasticum.
  
  Methods: Patients received baseline skin examinations, target skin lesion evaluation, and photography; renal function tests and serum calcium and phosphate levels; urine calcium, phosphate, and creatinine levels; skin biopsy; and eye examinations and indocyanine-green angiography. Patients were treated with aluminium hydroxide tablets or liquid and returned every 2 to 4 months for skin photography and lesion evaluation. Repeated skin biopsies were performed on clinically improved target sites. Ophthalmologic evaluation was obtained at yearly intervals.
  
  Results: Of six patients, three showed significant clinical improvement of skin lesions and all three of these patients showed histopathologic regression of disease in their target lesions. No deterioration of eye disease was seen in any of the six patients at one-year follow-up.
  
  Conclusion: Our results demonstrate that the calcification seen in pseudoxanthoma elasticum may be reversible in some patients. This could hold true for eye and vascular lesions and for skin. Further studies supporting these results could reveal the first real treatment option for PXE. ( J Am Acad Dermatol 2005;53:610-5.)
  
  Sherer DW, Singer, G, Uribarri J, Phelps RG, Sapadin AN, Freund KB, Yanuzzi L, Fuchs W, *Lebwohl M, New York, New York, USA
   

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• Pediatric study
  Ludovic Martin, Dept. of Dermatology, Angers University Hospital, Angers, France
  

   

  The diagnosis of pseudoxanthoma elasticum (PXE) is most often made late in the second or in the third decade of life. We aimed to describe the manifestations of PXE before the age of 15 years in our cohort of 96 patients (2001 – 2007) (Br J Dermatol 2009). Children under age 15 years with definite PXE and adults in whom serious manifestations of PXE had occurred before the age of 15 years were evaluated in our referral center. Fifteen patients (16%) had pediatric onset of the disease. Nine children were diagnosed at a mean age of 10 years. Cutaneous lesions were the presenting symptom in eight. None had cardiovascular or ophthalmological symptoms. Six adult patients had had severe cutaneous and/or cardiovascular (angina pectoris, intermittent claudication, GI hemorrhage) manifestations before the age of 15 years. Both adult patients with early extensive skin lesions had the PXE-like condition related to the GGCX gene.
  
  Absence of complications is common in children, but severe complications are unpredictable. Early diagnosis of PXE may be important to provide accurate information and discuss lifestyle adjustments in order to improve the prognosis of pseudoxanthoma elasticum.
  
  Our evaluation of pediatric PXE was also useful for further comprehensive steps in the pathophysiology of PXE. Recently, we described an unusual severe cardiovascular case of pseudoxanthoma elasticum presenting as generalized arterial calcification of infancy, a condition usually linked to ENPP1 gene sequence changes (Am J Med Genet 2010). This report demonstrated that PXE may be very severe in the neonate. Then, a collaborative work further demonstrated that, conversely, ENPP1 may be involved in some cases of pediatric PXE in the absence of ABCC6 mutations. This emphasized that ENPP1 and ABCC6 products are probably involved in the same molecular pathway that results in aberrant mineralization (ASHG, 60th annual meeting).
   

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• Ethical issues in predictive genetic testing for late onset disorders in children
  Lionel Bercovitch*, M.D., Department of Dermatology, Hasbro Children's Hospital and Alpert Medical School, Brown University, Providence, RI and PXE International, Washington, DC
  

   

  Predictive genetic testing includes presymptomatic testing which identifies those children with a near 100% chance of developing a specific genetic disorder, and predispositional testing which identifies the presence of mutations which predispose to the development of an illness. Most guidelines suggest that predictive testing for late onset disorders be delayed until the earliest age at which either medical surveillance is appropriate or at which symptoms develop, or until the child is old enough to give informed, competent assent as an adolescent or is legally able to give informed consent. Most geneticists support early presymptomatic testing for disorders that have effective early intervention but not for those disorders for which no intervention or treatment is available. The relevant ethical principles as well as the risks and benefits of presymptomatic testing are reviewed. The potential role and limitations of presymptomatic testing for pseudoxanthoma elasticum (PXE) are reviewed, as is the experience to date with presymptomatic testing for PXE since it has become commercially available in the US.
   

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