2012 PXE Research Meeting
The principal researchers working on PXE from laboratories around the world convened in Bethesda, Maryland, USA, on September 24-25, 2012 to participate in the 2012 PXE Research Meeting organized by PXE International, the premier lay organization advocating on behalf of PXE patients and their families. The presentations and ensuing discussions provided state-of-the-art, up-to-date information on epidemiology, diagnostics, pathomechanisms, and treatment potential relating to PXE, with translational implications. Further research, including identification of specific molecules transported by ABCC6, is necessary for development of global, pathophysiology-related approaches to treat this currently intractable disorder.
Click here: Summary article in Journal of Investigative Dermatology
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 Jun;133(6):1444-9.
Click here: Lay summary of the 2012 PXE Research Meeting
Below find presentations abstracts from the 2012 PXE Research Meeting. Click to expand or hide.
- ENPP1 mutations in PXE and ABCC6 mutations in GACI - the phenotypic overlap
Jouni Uitto, Jefferson Medical College, Philadelphia, PAQiaoli Li, Jouni Uitto*
Jefferson Medical College, Philadelphia, PA
A number of Mendelian genetic disorders manifest with ectopic mineralization, particularly affecting the skin and the cardiovascular system. The prototype of such diseases is pseudoxanthoma elasticum (PXE), a multi-system disorder manifesting primarily in the skin, the eyes, and the cardiovascular system. The classic PXE is caused by mutations in the ABCC6 gene. Generalized arterial calcification of infancy (GACI), an autosomal recessive disorder, is characterized by congenital calcification of large and medium sized arteries and periarterial connective tissues, leading to early myocardial infarction and stroke and often to death during infancy or childhood. Early on, most patients with GACI were shown to harbor inactivating mutations in the ENPP1 gene. Recent observations by us and others have indicated a phenotypic overlap between PXE and GACI. First, we examined a two-year old patient with extensive tissue mineralization, including vascular calcification of both forearms, and calcification on the right atrium, left circumflex artery and left main coronary artery. Skin examination revealed yellowish papules coalescing into cobblestone plaques distributed circumferentially on the neck, inguinal folds and abdomen, suggesting the diagnosis of PXE (1). Skin biopsy revealed clumped, irregular elastic fibers in the reticular dermis depicting mineralization. Based on these observations, the clinical diagnosis of PXE was made; however, no pathogenetic mutations were found in ABCC6. In contrast, sequencing of ENPP1 revealed a homozygous sequence variant, c.1538A→G (p.Y513C), a pathogenetic mutation that has been previously reported in an Italian patient with GACI, in trans with p.Y659C mutation. Thus, mutations in the ENPP1 gene can be associated with cutaneous findings of PXE. In a second set of studies, we examined 6 families with GACI, in which initial screening for ENPP1 did not disclose pathogenetic mutations. In four of these families either homozygous or compound heterozygous mutations in both alleles of the ABCC6 gene were discovered, and two families carried a heterozygous mutation in ABCC6. Thus, mutations in the ABCC6 gene can be associated, in addition to PXE, also with GACI (2, 3). In addition to ABCC6 and ENPP1, a number of other genes can harbor mutations resulting in ectopic mineralization, including SAMD9 in normophosphatemic familial tumoral calcinosis, as well as GALNT3, FGF23 and KL genes in the hyperphosphatemic variants (4). In addition, patients with CD73 deficiency, due to mutations in the NT5E gene, depict vascular mineralization, clinically similar but histopathologically distinct from PXE (5). Collectively, these observations support the notion of an intricate mineralization/anti-mineralization network in tissues, and careful balance of factors both promoting and preventing connective tissue mineralization at the local level is required for normal homeostasis.
1. Li et al., Br. J. Dermatol. 166:1107-1111, 2012
2. Brodsky et al., unpublished
3. Nitschke et al., Am. J. Hum. Genet 90:25-39, 2011
4. Sprecher, J. Invest. Dermatol. 130:652-660, 2010
5. Markello et al., Mol. Genet. Metab. 103:44-50, 2011
- PXE/cutis laxa - clinical overlap
Olivier Vanakker, Ghent University Hospital, Ghent, BelgiumAbstract not available.
- New insights into the pathogenesis of pseudoxanthoma elasticum by identifying genetic interactions and modifiers
Doris Hendig, Institute of Laboratory and Transfusion Medicine of the Heart and Diabetes Center, Bad Oeynhausen, GermanyHendig, D.*1, Dabisch-Ruthe, M.1, Prior, K.K.1, Kuzaj, P.1, Götting, C.1, Szliska, C.2, Charbel-Issa, P.3, Knabbe, C.1
1Herz- und Diabeteszentrum NRW, Universitätsklinik der Ruhr-Universität Bochum, Institut für Laboratoriums- und Transfusionsmedizin, Bad Oeynhausen, Germany
2Krankenhaus Bethesda, Lehrkrankenhaus der Ruhr-Universität Bochum, Dermatologie, Freudenberg, Germany
3Universitätsaugenklinik Bonn, Bonn, Germany
Screening of the ATP binding cassette transporter protein subfamily C member 6 gene (ABCC6) in 97 German Pseudoxanthoma elasticum (PXE) patients identified mutations in 168 of 194 alleles, revealing a mutation detection rate of 87%. This is in accordance with other studies showing that 5-15% of PXE patients lack one or both ABCC6 mutations. Although it was shown that approximately 25% of the unidentified disease alleles underlie larger deletions/insertions not detectable by standard sequencing technologies, there remain several PXE patients with no clear genotype. The recent identification of a PXE-like syndrome caused by a different genetic defect and other PXE-related diseases (e.g. GACI) points to genetic interactions between ABCC6 and other candidate genes. Moreover, high intra-familiar and inter-individual clinical variability of PXE patients and the lack of genotype-phenotype correlations, leads to the assumption that secondary genetic cofactors exist. The aim of our study was to investigate selected functional polymorphisms in candidate genes, encoding gene products known to act in regulation of matrix calcification, degradation and angiogenesis, in a large cohort of German PXE patients and age- and sex-matched healthy controls by DHPLC-, RFLP-, allele-specific PCR, and direct sequencing. We identified several functional polymorphisms found to be statistically associated with PXE manifestation (SPP1, ENPP1, VKORC1), PXE severity (VEGFA) and course of disease (different antioxidant genes). One haplotype arising from two promoter polymorphisms c.-7G>A and c.-138T>C in the MGP gene, encoding for the local calcification inhibitor matrix Gla protein, was more frequently detected in the healthy control cohort, and therefore postulated as a potential protective co-factor in PXE manifestation. Our findings add significant support to the role of modifier genes contributing to the aetiopathology of PXE and underscore the importance of analyzing gene-gene-environment interactions for understanding the pathogenesis of complex - even if monogenic - phenotypes such as PXE.
- Next-gen sequencing of ABCC6 and other PXE and modifying genes
Michael Dean, National Cancer Institute, NIH, Frederick, MDMichael Dean*1, Michal Dyba1, Alana Ebert-Zavos1, Sevilay Turan1, Alexander Borsa1, Sharon Terry2, and Karobi Moitra1
1National Cancer Institute, NIH, Frederick, MD
2PXE International, Inc., Washington, DC
Pseudoxanthoma elasticum (PXE) is a rare autosomal recessive disorder, which causes elastic tissue in the body to mineralize. PXE primarily manifests in the skin, eyes, cardiovascular system, and gastrointestinal system and impacts 1 in 25,000 people to 1 in 100,000 people worldwide. Currently there are no known treatments for PXE, however it is primarily caused by mutations in the ABCC6 gene. The trans-membrane protein ABCC6 is encoded by this gene, and serves as a transporter primarily expressed in the liver and kidneys. PXE has a high phenotypic variability with over 200 documented mutations discovered so far. By analyzing the genetic makeup of PXE patients using NextGen sequencing approaches, we have recently discovered 2 previously undiscovered novel mutations in the ABCC6 gene and have discovered the presence of mutations in patients which were previously undetected by DHPLC (Denaturing high performance liquid chromatography) suggesting that NextGen sequencing technology for specific genes could be a viable alternative to PCR and DHPLC in clinical diagnostics. From the discovery of these mutations, correlations can be formed between an individual’s genetic makeup and the specific manifestations of PXE in that individual. By using this new information about the genetics of PXE and correlating it to the available clinical data we can identify how amino acid variability affects the ABCC6 protein and thus, the disease itself. The targeted sequencing array also contained primers for the SOD2, MGP, MMP2, GGCX and other candidate genes, and the characterization of variants in these genes may also shed light on PXE pathology.
- Warfarin accelerates the ectopic mineralization in Abcc6-/- mice - clinical relevance to pseudoxanthoma elasticum
Qiaoli Li, Jefferson Medical College, Philadelphia, PAQiaoli Li*1, Haitao Guo1, David Chou1, Dominique Harrington2, Leon J. Schurgers2, Sharon F. Terry3 and Jouni Uitto1
1Jefferson Medical College, Philadelphia, PA
2Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands
3PXE International, Inc., Washington, DC
Pseudoxanthoma elasticum (PXE), an autosomal recessive disorder caused by mutations in the ABCC6 gene, manifests with ectopic mineralization of peripheral connective tissues with clinical findings in the skin, the eyes and the cardiovascular system. Warfarin, a commonly used anticoagulant, is associated with increased mineralization of the arterial blood vessels and cardiac valves. We hypothesized that warfarin may accelerate the ectopic tissue mineralization in PXE, with clinical consequences. To test this hypothesis we developed a model in which Abcc6-/- mice, which recapitulate features of PXE, were fed with diet supplemented with warfarin and vitamin K1. Inclusion of vitamin K1 in the diet prevents the mice from developing a bleeding disorder. Warfarin clearly interfered with the vitamin K cycle, as demonstrated by significantly increased levels of vitamin KO (epoxide). Examination of the mice placed on warfarin-containing diet by quantitative chemical and morphometric analyses revealed massive accumulation of mineral deposits, which were shown to consist of hydroxyapatite, in the dermal sheath of vibrissae, kidneys, heart, aorta and eyes. Previous studies have suggested that matrix Gla protein (MGP), a powerful anti-mineralization factor, plays a role in PXE. Consequently, our results suggest that reduction in the γ-glutamyl carboxylation of MGP as a result of warfarin treatment allowed progressive tissue mineralization to ensue. To explore the clinical relevance of these findings, the PXE International database, consisting of ~4,000 patients with PXE, was queried about the use of warfarin. Of the respondents, 2.6% reported past or present use of warfarin for the average length of 4.1 ± 3.4 years. Based on the estimated prevalence of PXE, ~1:50,000, thousands of patients with PXE globally may be at risk for increased ectopic mineralization and severity of PXE as a result of warfarin therapy.
- Dietary magnesium/phosphate influences carotid intima-media thickness - a novel biomarker for PXE
Erine A. Kupetsky, University of Pittsburgh Medical Center, Pittsburgh, PAKupetsky, EA*, Li, Q, Uitto, J.
Dept. of Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, PA
Pseudoxanthoma elasticum (PXE), which demonstrates progressive build-up of calcium phosphate and proteoglycan deposits in skin, eye, and arteries, has been associated with myocardial infarctions, stroke, and blindness. In a mouse model of PXE, a magnesium-enriched diet prevents mineralization of the vibrissae capsule, an early biomarker for PXE. However, biomarkers for therapeutic responses in PXE have not been identified in humans. Since PXE patients have an increased carotid intima-media thickness (CIMT), a risk factor for cardiovascular disease and stroke, we analyzed the feasibility of CIMT as a treatment endpoint before and after magnesium supplementation in a mouse model of PXE (Abcc6-/-). CIMT was measured in one year-old Abcc6-/- and Abcc6+/+ mice fed either standard rodent diet with or without magnesium oxide supplementation for two months. Baseline CIMT in Abcc6 -/- vs. Abcc6+/+ mice was increased (p-value = 0.009), while CIMT in magnesium-treated vs. untreated Abcc6-/- mice was reduced (p-value = 0.024). CIMT is a novel treatment endpoint in this mouse model and may serve as a predictive biomarker of therapeutic response in PXE patients. In that context, magnesium oxide significantly reduced CIMT in PXE mice, and may be useful for disease prevention in PXE patients.
- Beyond the concept of defective carboxylation process in PXE
Daniela Quaglino, University of Modena and Reggio Emilia, Modena, ItalyQuaglino D.*, Boraldi F., Annovi G., Costa S., § Schurgers L., # Vermeer C
Dept. of Life Sciences, University of Modena and Reggio Emilia, Modena (Italy)
§ Dept. of Biochemistry, University of Maastricht, Maastricht (The Netherlands)
# VitaK, University of Maastricht, Bio Partner Center, Maastricht (The Netherlands)
Introduction and aim: Mineralization of elastic fibers in Pseudoxanthoma elasticum (PXE) has been associated with low levels of carboxylated Matrix Gla Protein (MGP), most likely as a consequence of reduced vitamin K availability. Unexpectedly, vitamin K supplementation does not exert beneficial effects on soft connective tissue mineralization in the PXE animal model. Key question is whether PXE fibroblasts are able to utilize vitamin K and to restore an adequate MGP carboxylation. We have therefore performed in vitro studies for a direct investigation of the response of human peripheral mesenchymal cells, avoiding interferences due to dietary absorption properties and/or transport capabilities in the circulation.
Materials and Methods: Dermal fibroblasts from control subjects and from PXE patients were cultured in the presence of vitamin K1 and K2 in standard conditions and in a calcifying medium (supplemented with b-glycerophosphate, dexamethasone and ascorbic acid). Intracellular accumulation of vitamin K, total and MGP carboxylation, and in vitro calcification have been evaluated.
Results: Control and PXE fibroblasts were characterized by a similar dose-dependent uptake of both vitamin K1 and K2, thus promoting in all cell lines a significant increase of total protein carboxylation. Nevertheless, MGP carboxylation remained much less in PXE fibroblasts. Consistently, the mineralization process induced in vitro by a long-term culture in a calcifying medium appeared unaffected by vitamin K.
Conclusions: Results provide new evidence excluding the hypothesis that PXE fibroblasts are not capable to utilize vitamin K and to up-regulate the carboxylation process, and suggest that altered MGP characteristics/properties could contribute to defective carboxylation, even at increased levels of vitamin K.
Work supported by FCRMO (EctoCal) and PXE International.
- Microcomputed tomography for quantitation of mineralization in Abcc6-/- mice
Olivier Le Saux, University of Hawaii, Honolulu, HIOlivier Le Sauxa,b, Yannick Le Correa, Florence Froeligera, Hélène Liboubanc, Serge Willoteauxa, Georges Lefthériotisa, and Ludovic Martina
aL'UNAM university, Integrated Neurovascular and Mitochondrial Biology (BNMI), UMR CNRS 6214 / INSERM 771, Angers School of Medicine, Angers, France
bDepartment of Cell and Molecular Biology; John A. Burns School of Medicine, University of Hawai’i, Honolulu, HI
cUniversity of Angers, INSERM, U 922 "Remodelage osseux et Biomatériaux", Angers School of Medicine, Angers, France
Pseudoxanthoma elasticum (PXE) in humans and dystrophic cardiac calcification (DCC) in mice are heritable disorders characterized by dystrophic calcification of soft connective tissues and related to the defective function of ABCC6 (human) /Abcc6 (mouse) transporter. Of particular interest is the finding of calcified vibrissae in Abcc6-/- mice, that facilitate the study of dystrophic calcification by histological techniques. We aimed at determining if mice prone to DCC (C3H/HeOuJ and DBA/2J strains) presented similar vibrissae changes, and at evaluating the value of microcomputed tomography (microCT) to quantify the extent of mystacial vibrissae calcifications. These calcifications were absent in DBA/2J and control C57BL/6J mice. In both Abcc6-/- and C3H/HeOuJ mice, calcifications progressed in a caudal-rostral direction with aging. However, the calcification process was delayed in C3H/HeOuJ mice indicating an incomplete penetrance of the calcification phenotype. We also found that the calcification process in the cephalic region was not limited to mystacial vibrissae, but was also present in other periorbital sensorial vibrissae. The vibrissae calcification was circular in shape and encompassed the medial region of the vibrissae capsule adjacent to the ring and cavernous sinuses, i.e. the areas adjacent to blood and lymphatic vessels. Collectively, our findings confirmed that Abcc6 acts as an inhibitor of spontaneous chronic mineralization and that microCT is a valuable non-invasive tool for the quantification and follow-up of the calcification phenotype in Abcc6-deficient mice.
- Experimental strategies to analyse missense disease-causing mutations of ABCC6 in vitro and in vivo
András Váradi, Hungarian Academy of Sciences, Budapest, HungaryViola Pomozi1, Christopher N. Brampton2, Qiaoli Li3, Jouni Uitto3, Olivier Le Saux2 and András Váradi*1
1Institute of Enzymology, RCNS, Hungarian Academy of Sciences, Budapest
2Dept. of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawaii
3Dept. of Dermatology and Cutaneous Biology, Jefferson Medical College, Philadelphia
The ABCC6 protein is an active organic anion transporter localized in the plasma membrane. We have embarked upon a systematic study to investigate the impact of missense disease-causing mutation in order to better understand the molecular failure triggered by different mutations. Ten mutant proteins were selected based on frequency in the PXE patient cohort and their position in the 3D homology model. The biochemical transport properties were determined by in vitro transport of SF9 membrane vesicles, the cellular localization of each variant were determined in vitro in polarized and non-polarized MDCKII cells as well as in vivo in mouse liver utilizing hydrodynamic tail vein injection. We have found that most of the disease-causing mutations result in impaired cellular processing and intracellular localization leaving the transport function unharmed. These mutants are good candidates for pharmacological correction. We have concluded that the in vitro localization methods cannot replace the in vivo approach, the closest to the physiology of the human liver.
Recently, the plasma membrane localization of ABCC6/Abcc6 has been questioned. We provide experimental data supporting that the protein is in the plasma membrane.
Silencing of the Abcc6a gene of zebrafish triggered a developmental phenotype, which could be rescued when the mRNA of human ABCC6 was also injected into the embryos. Consequently, zebrafish provides an in vivo model for investigating mutant form of ABCC6. Indeed, we have established that the disease-causing mutants fail to rescue the phenotype, irrespective whether they are transport- or localization mutants. Interestingly, one mutant (V1298F), which was found to be correctly situated in the plasma membrane (both in cell culture and in vivo in mouse liver), but showing minimal residual activity in the transport assay gave a partial (30%) rescue in the zebrafish experimental model.
- ABCC6 expression is regulated by a primate-specific sequence located in the first intron of the gene
Tamás Arányi, Hungarian Academy of Sciences, Budapest, Hungary
Hugues de Boussac1, Marcin Ratajewski2, Iwona Sachrajda2, Caroline Bacquet1, Tünde Kovács3, András Váradi1, Lukasz Pulaski2, Tamás Arányi1
1 Institute of Enzymology, Hungarian Academy of Sciences, Budapest, Hungary
2 Laboratory of Transcriptional Regulation, Institute of Medical Biology, Polish Academy of Sciences, Lodz, Poland
3 Membrane Biology Research Group, Semmelweis University, Hungarian Academy of Sciences, Budapest, Hungary
Purpose: In the present study we investigated the cell-type specific transcriptional regulation of the ABCC6 gene.
Methods: We investigated the transcriptional regulation of the gene, using DNase I hypersensitivity assay followed by luciferase reporter gene assays and chromatin immunoprecipitation (ChIP).
Results: We identified DNase I hypersensitive sites (HS) specific to cell lines expressing ABCC6. These sites are located in the proximal promoter and in the first intron of the gene. We observed Hepatocyte Nuclear Factor 4α (HNF4α) and CCAAT/Enhancer binding protein ß (C/EBPß) binding to the proximal promoter and the primate-specific second intronic HS, respectively. We also showed that C/EBPß interacts with the proximal promoter of the gene and propose that it forms a complex with other regulatory proteins including the previously identified regulatory factor HNF4α.
Conclusions: Our data indicate that this complex, accounts for the tissue and primate-specific expression of the gene.
- Gender differences in pseudoxanthoma elasticum: Insights from the 2012 PXE Epidemiological Study
Lionel Bercovitch, Brown University, Providence, RILisa Ratanaprasatporn, ScB2, Juliann Reardon, BS2, Allison Chen, ScB2, Abbie Moore, PhD1, Sharon F Terry, MA1, Lionel Bercovitch, MD*1,2
1PXE International, Washington, DC
2Warren Alpert Medical School, Brown University, Providence, RI
OBJECTIVE: To study the differences in PXE phenotype, clinical course, and complications between males and females.
METHODS: 393 females and 120 males affected by PXE completed an online questionnaire. The instrument included questions about cutaneous, ocular, cardiac, gastrointestinal, and vascular signs and symptoms, as well as age at presentation and diagnosis, past treatments, and family history.
RESULTS: Females were disproportionately represented in the database, among respondents, and affected siblings, but females comprised a significantly larger portion of respondents than of their affected siblings. Females are more likely to have skin findings in all sites affected by PXE. Males tend to have more severe vision loss, experience retinal bleeding earlier, and are more likely to be legally blind. Males are significantly more likely to have angina and require coronary bypass surgery or angioplasty. They had significantly more hyperlipidemia and a higher mean BMI. There was no significant difference in reported rates of peripheral vascular disease.
LIMITATIONS: The response rate was low. The data are self-reported, rather than based on physical examination, which affects primarily physical signs, although it should affect males and females equally.
CONCLUSIONS: Although PXE appears to disproportionately affect females, some, but not all of this difference can be attributed to self-selection bias. There are significant differences in the severity of the cutaneous, cardiovascular, and ocular phenotypes between males and females.
REFLECTIONS: The apparent disproportion in number of females and males that are affected remains unexplained. It is not explained by simple Mendelian autosomal recessive inheritance. A larger sample of affected siblings might be more likely to establish if this is real or not. A better understanding of factors that account for the differences in severity of the ocular and cardiovascular phenotypes in males and females might be of some value in devising strategies for prevention of complications.
- The vascular calcification phenotype in pseudoxanthoma elasticum: Report from the French cohort
Georges Lefthériotis, Angers University Hospital, Angers, FranceLefthériotis G*1, Kauffenstein G2
1Dept of Vascular Investigations, University Hospital of Angers, France
2UMR Inserm 1083/CNRS 6214, Medical School Angers, France
Vascular calcification is one of the common phenotypical manifestations reported in pseudoxanthoma elasticum (PXE) patients. The mineralization that is typically associated with elastic fiber fragmentation primarily preferentially affects the medial layers of medium and small-sized muscular peripheral arteries leading to a medial elastino-calcinosis. Although the main clinical consequences of the arterial disease of PXE are characterized by increased risks for coronary, cerebral and lower limb arterial diseases, the fine structural and functional changes occurring in the arterial wall of PXE patients are still not understood. Data on the vascular phenotype of a PXE cohort of French origin have shown that PXE-specific arterial remodeling and calcification do not increase arterial stiffness, contrary to other calcifying arterial acquired metabolic disorders such as the Monckeberg's disease. These results suggested that the mineralization in PXE might not be the sole pathological determinant of the clinical presentation of PXE patients.
- An altered lymphatic vasculature contributes to the calcification phenotype in pseudoxanthoma elasticum
Olivier Le Saux, University of Hawaii, Honolulu, HIYannick Le Corre1,2, Christopher N. Brampton2, Oana Bollt2, Marjolijn Renard3, Olivier M. Vanakker3, Anne De Paepe3, Isabelle Quéré4, Georges Lefthériotis1, Ludovic Martin1 and Olivier Le Saux*2
1: LUNAM University, INMB, UMR CNRS 6214 / INSERM 1083, Angers School of Medicine, Angers, France
2: Department of Cell and Molecular Biology; John A. Burns School of Medicine, University of Hawai’i, Honolulu, HI
3: Center for Medical Genetics, Ghent University Hospital; Ghent, Belgium
4: Department of vascular medicine, Montpellier University Hospital, Montpellier, France
Pseudoxanthoma elasticum (PXE) is an inherited disease characterized by dystrophic calcification in dermal, vascular and ocular tissues. The current consensus is that the molecular pathology of PXE rests with the inability of the liver (because of inactivating ABCC6 mutations) to excrete an unidentified substrate(s) that otherwise prevents calcification in peripheral connective tissues. As we have observed a correlation between the PXE cutaneous manifestations and the regions of the sub-dermal lymph nodes, we hypothesized that lymphatic vessels and nodes could drain, concentrate and/or be affected by a PXE-specific factor(s). We have evaluated this hypothesis with Abcc6-/- mice and found significant arterial and dermal calcification in axillar, mesenteric, cervical, lumbar and inguinal lymph node areas as well as within the cortex of certain nodes. These manifestations were associated with an expansion of the lymphatic vessel network in the dermis of Abcc6-/- mice. No difference in lymphatic drainage in the skin was measured but dye extravasation under inflammatory conditions was decreased in older mice suggesting a PXE-specific adaptive response. Our results showed that areas of high lymphatic density, that include the vibrissae, correlated with significant calcification in Abcc6-/- mice suggesting that the lymphatic vasculature is affected and/or contribute to the pathophysiology of PXE.
- A novel mouse model for PXE: The KK/HlJ mouse
Annerose Berndt, University of Pittsburgh School of Medicine, Pittsburgh, PA
Berndt A*1, Li Q2, Uitto J2, and Sundberg JP3
1University of Pittsburgh, Pittsburgh, PA
2Thomas Jefferson University, Philadelphia, PA
3The Jackson Laboratory, Bar Harbor, ME
The B6.129-Abcc6tm1Jfk and other targeted mutant mice for this gene revealed that mineralization of the vibrissa sheath was potentially a pathognomonic lesion for PXE in mice. In a massive phenotyping study of 31 aging inbred strains of mice, the KK/HlJ inbred strain was found to have this lesion as well as systemic mineralized lesions similar to those found in the Abcc6 targeted mutant mice. Haplotype analysis of the strains under investigation revealed that several other strains in this study also had the same mutated Abcc6 allele. Of these, only the 129S1/SvlmJ strain had vibrissa mineralization and only in very old mice. Many of the investigated strains had cardiac mineralization and fibrosis, which has been associated with this same allelic mutation in C3H/HeJ and other strains. However, similar lesions were found in strains with the wild-type Abcc6 gene suggesting the heart lesions are not primary aspects of PXE in mice but that the mutated Abcc6 allele may act more as a modifier gene. In addition, KK/HlJ mice have a severe bone lesion called fibro-osseous disease. While this was most severe and appeared earlier in KK/HlJ mice than the other strains, the bone lesions were found in mice both with and without the Abcc6 mutation, again suggesting that Abcc6 acts as a modifier, not a primary gene, in this disease process. At least 5 other genes mutated in mice result in phenotypes very similar to the mouse PXE phenotype suggesting they may be other genes to investigate in humans as either primary or modifier genes. The KK/HlJ mice exhibit the most severe form of mouse PXE and when mated with other strains with the same Abcc6 allelic mutation these genetic studies will provide an important new tool to identify modifier genes for PXE.
- Cell-based therapies: Bone marrow transplantation, iPSCs and MSCs
Qiujie Jiang, Jefferson Medical College, Philadelphia, PAQiujie Jiang*1, Shunsuke Takahagi1, Yong Zhou1, Ganna Bilousova2, Dian Wang1, Dennis Roop2, Jouni Uitto1
1Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Philadelphia, PA
2Department of Dermatology, and Charles C. Gates Center for Regenerative Medicine and Stem Cell Biology, University of Colorado, Aurora, CO
Aberrant mineralization of peripheral connective tissues is a characteristic feature of pseudoxanthoma elasticum (PXE), a heritable metabolic disorder caused by mutations in the ABCC6 gene. This gene, which encodes an ABC transmembrane efflux transporter, is expressed primarily in the liver. There is currently no effective treatment for PXE. In this study, we have investigated the potential of induced pluripotent stem cells (iPSCs), the whole bone marrow (BM), and bone marrow derived mesenchymal stem cells (MSCs) for liver regeneration, with the aim to rescue PXE phenotype in Abcc6-/- mice, a model system that recapitulates the genetic, histopathologic, and ultrastructural features of PXE. We characterized mouse fibroblast-derived iPSCs, BM and purified MSCs, which were derived from GFP+ transgenic mice, and demonstrated that iPSCs and MSCs types have the capability of differentiating into hepatic lineages, hepatoblasts and hepatocytes in vitro. We also demonstrated the capability of these cells home to the liver after administration through intravenous injection following partial hepatectomy, with or without low dose irradiation. Both Abcc6 and GFP positive engrafted cells were found in the liver by immunofluorescence at two months after transplantation. These therapeutic models provide a platform for translational application of cell-based liver reconstitution as a means of regenerative therapy for PXE.
- PTC read-through of ABCC6 nonsense mutations
Yong Zhou, Jefferson Medical College, Philadelphia, PAYong Zhou*, Shunsuke Takahagi, Qiujie Jiang, Jouni Uitto
Dept. of Dermatology and Cutaneous Biology, Jefferson Medical College, Philadelphia, PA
Pseudoxanthoma elasticum (PXE) is an autosomal recessive metabolic disorder with estimated prevalence of 1:50,000, but with no current effective treatment. PXE is caused by mutations in the ABCC6 gene, and more than 300 different mutations are known to cause PXE, more than 35% of these being nonsense mutations. In this study, we investigated the therapeutic potential of the premature termination codon (PTC) readthrough-inducing drug PTC 124 in treating PXE in an in vitro system. The ability of drug to readthrough PTC was examined in HEK293 cells transfected with seven different ABCC6 nonsense DNA-constructs linked to a DDK peptide tag at the 3’ end associated with PXE. The efficacy of the read-through was evaluated by immunofluorescence and In-Cell ELISA at ~ 72 hours after treatment with the drug. Our preliminary data demonstrated that PTC124 did not exhibit toxicity at concentrations up to 40 μg/ml and that the highest readthrough efficiency was observed at dosage of 5 μg/ml when a series of doses were tested with a range from 0 to 40 μg/ml. In addition, PTC124 at 5μg/ml was more efficient on R1164X and R1275X than on other mutants tested, including R1141X, indicating that induced readthrough is not only a dose-dependent but also a context-dependent process. Although numerous steps need to be established to achieve a clinical benefit by inducing suppression of nonsense mutations, this study provides valuable information concerning the potential of pharmacological treatment for PXE.
- Clinical trials with dietary modulation
Mark Lebwohl, Mt. Sinai School of Medicine, New York, NY
Abstract not available.