Heritable Ectopic Mineralization Disorders Research Articles

Commentary on Variants in the ABCC6 Gene Implicated in Pseudoxanthoma Elasticum, a Heritable Ectopic Mineralization Disorder

Significant progress has been made in understanding pseudoxanthoma elasticum (PXE), which results from mutations in ABCC6. The low prevalence of PXE and its heterotypic presentation confound genotype-phenotype correlations and the characterization of many identified variants. Kowal etal. (2022) present an invivo model to characterize and annotate ABCC6 variants, establishing a novel system for allele annotation in the patient population.

Letter by Spiering et al Regarding Article, “Effect of Denosumab or Alendronic Acid on the Progression of Aortic Stenosis: A Double-Blind Randomized Controlled Trial”

HomeCirculationVol. 144, No. 22Letter by Spiering et al Regarding Article, “Effect of Denosumab or Alendronic Acid on the Progression of Aortic Stenosis: A Double-Blind Randomized Controlled Trial” Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessLetterPDF/EPUBLetter by Spiering et al Regarding Article, “Effect of Denosumab or Alendronic Acid on the Progression of Aortic Stenosis: A Double-Blind Randomized Controlled Trial” Wilko Spiering, MD, PhD, Willem P. Mali, MD, PhD and Pim A. de Jong, MD, PhD Wilko SpieringWilko Spiering https://orcid.org/0000-0002-2493-6407 Departments of Vascular Medicine (W.S.), University Medical Center Utrecht, The Netherlands. Search for more papers by this author , Willem P. MaliWillem P. Mali Radiology (W.P.M., P.A.d.J.), University Medical Center Utrecht, The Netherlands. Search for more papers by this author and Pim A. de JongPim A. de Jong Radiology (W.P.M., P.A.d.J.), University Medical Center Utrecht, The Netherlands. Search for more papers by this author Originally published29 Nov 2021https://doi.org/10.1161/CIRCULATIONAHA.121.055622Circulation. 2021;144:e334To the Editor:We read with interest the article by Pawade et al1 on the effects of denosumab or alendronate on the progression of aortic stenosis. The authors conclude from this randomized, controlled trial that both drugs did not affect progression of aortic valve calcification in patients with calcific aortic stenosis. They call for a search to find alternative pathways and mechanisms to identify disease-modifying therapies for the growing population of patients with this potentially fatal condition. We think that this trial does not justify abandoning bisphosphonate therapy as a potential treatment, but that it would have been better if the authors had chosen etidronate as the comparator instead of alendronate.Mineralization in soft tissues such as arteries and valves is controlled by endogenous inhibitors of hydroxyapatite formation, of which inorganic pyrophosphate is the most important. Bisphosphonates are nonhydrolyzable analogs of inorganic pyrophosphate, and cumulating evidence shows that they can inhibit vascular calcification. Nonnitrogenous bisphosphonates, like etidronate, are potent inhibitors of ectopic calcification, whereas nitrogenous bisphosphonates, like alendronate, have a high affinity for bone and are more potent inhibitors of osteoclasts. In an ex vivo model of calcification in intact aortic valves, it was shown that etidronate completely prevented aortic valve leaflet calcification at already very low concentrations.2Pseudoxanthoma elasticum is a heritable ectopic mineralization disorder with increased medial arterial calcification. In an animal model of pseudoxanthoma elasticum, etidronate, but not alendronate, significantly reduced calcification, indicating that the inhibiting effect on ectopic calcification of bisphosphonates is not just a class effect.3 In a double-blind, placebo-controlled, randomized, controlled trial in patients with pseudoxanthoma elasticum, we showed that etidronate reduced arterial calcification.4 However, in a randomized, controlled trial in patients with chronic kidney disease, alendronate did not decrease the progression of vascular calcification.5We think that the absence of any effect of alendronate on the progression of aortic stenosis seen in the study of Pawade et al is in line with earlier findings that alendronate with its low affinity for soft tissues is not the ideal bisphosphonate for inhibiting ectopic calcification.We would like to know why, with cumulating evidence that etidronate is very efficacious in inhibiting soft tissue calcification, the authors decided to choose alendronate and not etidronate in their clinical trial.Article InformationDisclosuresNone.FootnotesCirculation is available at www.ahajournals.org/journal/circ

Genetic heterogeneity of heritable ectopic mineralization disorders in a large international cohort

PurposeHeritable ectopic mineralization disorders comprise a group of conditions with a broad range of clinical manifestations in nonskeletal connective tissues. We report the genetic findings from a large international cohort of 478 patients afflicted with ectopic mineralization. MethodsSequence variations were identified using a next-generation sequencing panel consisting of 29 genes reported in association with ectopic mineralization. The pathogenicity of select splicing and missense variants was analyzed in experimental systems in vitro and in vivo. ResultsA total of 872 variants of unknown significance as well as likely pathogenic and pathogenic variants were disclosed in 25 genes. A total of 159 distinct variants were identified in 425 patients in ABCC6, the gene responsible for pseudoxanthoma elasticum, a heritable multisystem ectopic mineralization disorder. The interpretation of variant pathogenicity relying on bioinformatic predictions did not provide a consensus. Our in vitro and in vivo functional assessment of 14 ABCC6 variants highlighted this dilemma and provided unambiguous interpretations to their pathogenicity. ConclusionThe results expand the ABCC6 variant repertoire, shed new light on the genetic heterogeneity of heritable ectopic mineralization disorders, and provide evidence that functional characterization in appropriate experimental systems is necessary to determine the pathogenicity of genetic variants.

Functional Assessment of Missense Variants in the ABCC6 Gene Implicated in Pseudoxanthoma Elasticum, a Heritable Ectopic Mineralization Disorder

Pseudoxanthoma elasticum, a heritable multisystem ectopic mineralization disorder, is caused by inactivating mutations in the ABCC6 gene. The encoded protein, ABCC6, a transmembrane transporter, has a specialized efflux function in hepatocytes by contributing to plasma levels of inorganic pyrophosphate, a potent inhibitor of mineralization in soft connective tissues. Reduced plasma inorganic pyrophosphate levels underlie the ectopic mineralization in pseudoxanthoma elasticum. In this study, we characterized the pathogenicity of three human ABCC6 missense variants using an adenovirus-mediated liver-specific ABCC6 transgene expression system in an Abcc6-/- mouse model of pseudoxanthoma elasticum. Variants p.L420V and p.R1064W were found benign because they had abundance and plasma membrane localization in hepatocytes similar to the wild-type human ABCC6 transgene, normalized plasma inorganic pyrophosphate levels, and prevented mineralization in the dermal sheath of vibrissae in muzzle skin, a phenotypic hallmark in the Abcc6-/- mice. In contrast, p.S400F was shown to be pathogenic because it failed to normalize plasma inorganic pyrophosphate levels and had no effect on ectopic mineralization despite its normal expression and proper localization in hepatocytes. These results showed that adenovirus-mediated hepatic ABCC6 expression in Abcc6-/- mice can provide a model system to effectively elucidate the multifaceted functional consequences of human ABCC6 missense variants identified in patients with pseudoxanthoma elasticum.

Therapeutics Development for Pseudoxanthoma Elasticum and Related Ectopic Mineralization Disorders: Update 2020.

Pseudoxanthoma elasticum (PXE), the prototype of heritable ectopic mineralization disorders, manifests with deposition of calcium hydroxyapatite crystals in the skin, eyes and arterial blood vessels. This autosomal recessive disorder, due to mutations in ABCC6, is usually diagnosed around the second decade of life. In the spectrum of heritable ectopic mineralization disorders are also generalized arterial calcification of infancy (GACI), with extremely severe arterial calcification diagnosed by prenatal ultrasound or perinatally, and arterial calcification due to CD73 deficiency (ACDC) manifesting with arterial and juxta-articular mineralization in the elderly; the latter disorders are caused by mutations in ENPP1 and NT5E, respectively. The unifying pathomechanistic feature in these three conditions is reduced plasma levels of inorganic pyrophosphate (PPi), a powerful endogenous inhibitor of ectopic mineralization. Several on-going attempts to develop treatments for these conditions, either with the goal to normalize PPi plasma levels or by means of preventing calcium hydroxyapatite deposition independent of PPi, are in advanced preclinical levels or in early clinical trials. This overview summarizes the prospects of treatment development for ectopic mineralization disorders, with PXE, GACI and ACDC as the target diseases, from the 2020 vantage point.

275 Permanent Alteration of Abcc6 With In Vivo CRISPR-Cas9 Genome Editing

Pseudoxanthoma elasticum (PXE), a heritable ectopic mineralization disorder, is caused by mutations in the Abcc6 gene primarily expressed in the liver and the kidneys. The fundamental question on the pathogenesis of PXE, whether lack of Abcc6 expression in liver or kidney is the primary site of molecular pathology in peripheral tissues, has not been addressed. Previous studies have used a systemic knockout to construct mice or zebrafish model, these animal models do not simulate the onset of PXE very well. Specific knock-out of the Abcc6 gene in the liver may better mimic the onset of PXE. Therefore, it is imperative to construct an animal model that specifically knocks out the ABCC6 gene in the liver. The goal of this study was to construct a liver-specific knockout animal model of Abcc6 gene, aiming to better simulate the pathogenesis of PXE and provide new ideas for its pathogenesis research and treatment. Our previous study found a new PXE site in Chinese, so we designed sgRNA at the same sites in mice and constructed a CRISPR/Cas9 plasmid co-expressing lentivirus. Then we used tail vein injection to achieve knock-out Abcc6 gene in the liver. We found that 1 month of administration of the virus, the mutagenesis of Abcc6 in the liver was found. We also found that 6 months of administration of the virus, pyrophosphate (PPi) level was reduced in the knock-out mice and depicted ectopic mineralization in the dermal sheath of vibrissae in muzzle skin. Gene editing with the CRISPR/Cas9 system disrupts the Abcc6 gene in vivo with high efficiency and reduces PPi levels in mice, and depicts ectopic mineralization in the skin. Specific knock-out of the Abcc6 gene in the liver may better mimic the onset of PXE. This approach may have therapeutic potential for the prevention of PXE in humans.

300 Assessment of Non-Canonical Splicing Variants in ABCC6 and Comparison with Bioinformatics Predictions

Pseudoxanthoma elasticum (PXE), a prototype of heritable ectopic mineralization disorders, is caused by inactivating mutations in the ABCC6 gene predominantly expressed in hepatocytes. Different types of mutations have been identified in ABCC6 including canonical splice site mutations affecting pre-mRNA splicing. Non-canonical splicing variants near or within exon-intron boundary in ABCC6 are under-recognized due to the lack of liver biopsies from patients carrying such variants, and consequently, inability to determine their consequences on splicing by transcriptional analysis. The interpretation of such variants has primarily relied on the prediction by in silico algorithms; often such programs do not provide consensus predictions. Among seven non-canonical splicing variants identified in ABCC6 in patients with PXE, five variants were found to affect splicing in in vitro minigene splicing assays in transfected HepG2 cells. Four of the variants, c.1868-5T>G, c.2070+5G>A, c.3735G>A (p.E1245=), and c.3883-46A>G, caused skipping of the respective adjacent exon. One variant, c.3883-6G>A, created a new splicing donor site resulting in a 4-bp retention from the intronic region. The results provide experimental evidence that these non-canonical variants disrupt splicing, suggesting the presence of pathogenic mutations beyond the canonical splice sites, including silent variants in the coding region.

405 Adenovirus-mediated ABCC6 gene therapy prevents ectopic mineralization in an Abcc6-/- mouse model of pseudoxanthoma elasticum

Biallelic loss-of-function mutations in the ABCC6 gene cause pseudoxanthoma elasticum (PXE) and type 2 generalized arterial calcification of infancy (GACI), heritable ectopic mineralization disorders without effective treatment. ABCC6 encodes the putative efflux transporter ABCC6 which is predominantly expressed in the liver. While the substrate of ABCC6 protein remains unknown, recent studies demonstrated that PXE is a metabolic disorder caused by reduced circulating levels of inorganic pyrophosphate (PPi), a potent inhibitor of mineralization. Here, we hypothesized that reconstitution of ABCC6 might counteract ectopic mineralization in an Abcc6-/- mouse model of PXE. Intravenous administration of a recombinant adenovirus expressing wild type human ABCC6 in Abcc6-/- mice showed sustained high-level expression of human ABCC6 in the liver for up to 4 weeks, increasing PPi levels in plasma. In addition, adenovirus injection every 4 weeks restored plasma PPi levels, and consequently, significantly reduced ectopic mineralization in the skin of young mice. By contrast, the same treatment in old mice, 43 weeks of age, with already established mineral deposits failed to reduce mineralization. These results suggest that adenovirus-mediated ABCC6 gene delivery, when initiated early, is a promising prevention therapy for PXE and GACI, diseases that currently lack preventive or therapeutic options to counteract ABCC6 deficiency.

406 An in vitro splicing assay reveals the pathogenicity of intronic variants in ABCC6, the gene at fault in pseudoxanthoma elasticum

Pseudoxanthoma elasticum (PXE), a prototype of heritable ectopic mineralization disorders, is caused by inactivating mutations in the ABCC6 gene predominantly expressed in hepatocytes. Different types of mutations have been identified in ABCC6 including canonical splice site mutations affecting pre-mRNA splicing. Splicing variants in ABCC6 are often overlooked due to the unavailability of liver biopsies from patients with such variants to determine their consequences on splicing by transcriptional analysis. Thus, the interpretation of intronic variants purely relies on computational algorithms to predict their effects on splicing. Very often the prediction programs do not provide a consensus as to the effect of variants for disrupting normal splicing. Our recent ABCC6 mutation analysis of over 400 patients with PXE shows that this continues to be a problem: among 7 intronic variants in ABCC6 with conflicting results by current splice site prediction algorithms, 3 variants were found to affect splicing. These three intronic variants were analyzed in an in vitro functional splicing assay using expression of minigene constructs in transfected HepG2 cells. Total RNA was extracted and RT-PCR was performed to analyze the splice pattern followed by Sanger sequencing. Two of the variants, c.3883-46A>G and c.2070+5G>A, caused skipping of the adjacent exon, predicted to cause out-of-frame translation and premature stop of translation. One variant, c.3883-6G>A, created a new splice acceptor site resulting in a 4-bp retention from the intronic region and causing frame-shift of translation. The results provided evidence that intronic variants away from the canonical splice sites disrupted splicing, clearly demonstrating the presence of mutations beyond the canonical splice sites.

Adenovirus-Mediated ABCC6 Gene Therapy for Heritable Ectopic Mineralization Disorders

Loss-of-function mutations in the ABCC6 gene cause pseudoxanthoma elasticum and type 2 generalized arterial calcification of infancy, heritable ectopic mineralization disorders without effective treatment. ABCC6 encodes the putative efflux transporter ABCC6, which is predominantly expressed in the liver. Although the substrate of ABCC6 remains unknown, recent studies showed that pseudoxanthoma elasticum is a metabolic disorder caused by reduced circulating levels of pyrophosphate, a potent mineralization inhibitor. We hypothesized that reconstitution of ABCC6 might counteract ectopic mineralization in an Abcc6-/- mouse model of pseudoxanthoma elasticum. Intravenous administration of a recombinant adenovirus expressing wild-type human ABCC6 in Abcc6-/- mice showed sustained high-level expression of human ABCC6 in the liver for up to 4 weeks, increasing pyrophosphate levels in plasma. In addition, adenovirus injection every 4 weeks restored plasma pyrophosphate levels and, consequently, significantly reduced ectopic mineralization in the skin of young mice. By contrast, the same treatment in old mice with already established mineral deposits failed to reduce mineralization. These results suggest that adenovirus-mediated ABCC6 gene delivery, when initiated early, is a promising prevention therapy for pseudoxanthoma elasticum and generalized arterial calcification of infancy, diseases that currently lack preventive or therapeutic options.

Heritable Ectopic Mineralization Disorders: Pathomechanisms and PotentialTreatment.

Pseudoxanthoma elasticum (PXE), a prototype of heritable ectopic mineralization disorders, is an autosomal recessive disease characterized by deposition of calcium hydroxyapatite in the skin, eyes, and cardiovascular system, with protean manifestations (Li etal., 2016; Li and Uitto, 2013; Neldner, 1988). The classic form of PXE is late-onset and slow-progressing, and the major clinical problems relate to loss of vision and development of cardiovascular complications (Neldner, 1988). The classic form of PXE is caused by loss-of-function mutations in the ABCC6 gene encoding ATP-binding cassette subfamily C, member 6 (ABCC6), a putative transmembrane efflux transporter protein expressed primarily in the liver and kidneys. The metabolic hypothesis concerning PXE postulates that the absence of functional ABCC6 activity, primarily in the liver, results in deficiency of circulating factor(s) that is physiologically required to prevent ectopic mineralization under normal calcium and phosphate homeostasis (Li etal., 2016) (Figure1). However, the factor(s) transported by ABCC6 from the intracellular milieu to the extracellular space have not been identified.

Inhibition of Tissue-Nonspecific Alkaline Phosphatase Attenuates Ectopic Mineralization in the Abcc6–/– Mouse Model of PXE but Not in the Enpp1 Mutant Mouse Models of GACI

Pseudoxanthoma elasticum (PXE), a prototype of heritable ectopic mineralization disorders, is caused by mutations in the ABCC6 gene encoding a putative efflux transporter ABCC6. It was recently shown that the absence of ABCC6-mediated adenosine triphosphate release from the liver and, consequently, reduced inorganic pyrophosphate levels underlie the pathogenesis of PXE. Given that tissue-nonspecific alkaline phosphatase (TNAP), encoded by ALPL, is the enzyme responsible for degrading inorganic pyrophosphate, we hypothesized that reducing TNAP levels either by genetic or pharmacological means would lead to amelioration of the ectopic mineralization phenotype in the Abcc6-/- mouse model of PXE. Thus, we bred Abcc6-/- mice to heterozygous Alpl+/- mice that display approximately 50% plasma TNAP activity. The Abcc6-/-Alpl+/- double-mutant mice showed 52% reduction of mineralization in the muzzle skin compared with the Abcc6-/-Alpl+/+ mice. Subsequently, oral administration of SBI-425, a small molecule inhibitor of TNAP, resulted in 61% reduction of plasma TNAP activity and 58% reduction of mineralization in the muzzle skin of Abcc6-/- mice. By contrast, SBI-425 treatment of Enpp1 mutant mice, another model of ectopic mineralization associated with reduced inorganic pyrophosphate, failed to reduce muzzle skin mineralization. These results suggest that inhibition of TNAP might provide a promising treatment strategy for PXE, a currently intractable disease.

Generalized arterial calcification of infancy with a novel ENPP1 mutation: a case report

BackgroundGeneralized Arterial Calcification of Infancy (GACI) is a heritable ectopic mineralization disorder resulting in diffuse arterial calcifications and/or stenosis, mostly caused by mutations in the ENPP1 gene. Here we present a case report of GACI in a male infant with a new familial mutation of the ENPP1 gene and the clinical outcome after biphosphonates therapy.Case presentationThe clinical presentation was characterized by a severe early-onset of hypertension refractory to multiple therapy. To investigate this atypical hypertension, a renal Doppler ultra-sonography was performed and diffuse echo-bright arteries were detected; then a low-dose whole-body computed tomography demonstrated extensive arterial calcifications, suggesting GACI. A novel homozygous mutation c.784A > G (p.Ser262Gly) was detected in the ENPP1 gene. The infant was administered four courses of bisphosphonates: arterial calcifications were found to decrease but severe refractory hypertension was persistent. Although GACI can be a rapidly fatal illness and frequently results in death in infancy, the patient was 24 months of age at the time of writing this report.ConclusionsThree points of interest: the first one is to remind clinicians of this rare and atypical etiology in neonates with severe hypertension and in fetuses with cardiomyopathy and non-immune hydrops fetalis. The second point is the identification of a novel mutation in the ENPP1 gene associated with a clinical presentation of GACI. The third point is the fairly favourable outcome of our patient after bisphosphonates therapy, with calcifications regression but not hypertension.

208 Targeting plasma pyrophosphate deficiency in mouse models of heritable ectopic mineralization disorders – PXE and GACI

Pathologic mineralization of the skin and vascular connective tissues is the hallmark of certain single gene heritable disorders. Pseudoxanthoma elasticum (PXE), caused in most cases by loss-of-function mutations in the ABCC6 gene, serves as a prototype of heritable multi-system ectopic mineralization disorders with normal calcium and phosphate homeostasis. Generalized arterial calcification of infancy (GACI), caused in most cases by mutations in the ENPP1 gene, has overlapping phenotypic features with PXE. However, it was recently shown that PXE and GACI in some cases can also be caused by mutations in either ENPP1 or ABCC6, respectively. These observations suggested the possibility of shared pathomechanistic pathways for these conditions, and specifically, studies in these diseases and their corresponding mouse models revealed reduced plasma levels of inorganic pyrophosphate, a powerful inhibitor of mineralization. Consequently, the results suggested that insufficient inorganic pyrophosphate in the blood circulation underlies ectopic mineralization in both conditions. Towards development of treatment for these ectopic mineralization disorders, we have performed studies targeting plasma pyrophosphate deficiency through direct supplementation of inorganic pyrophosphate, or administration of stable, nonhydrolyzable pyrophosphate analogues, bisphosphonates, for treatment of these ectopic mineralization disorders in mouse models of PXE and GACI, Abcc6-/- and Enpp1asj. These approaches resulted in reduced ectopic mineralization in these mice, suggesting novel treatment modalities for these currently intractable diseases.

Plasma PPi Deficiency Is the Major, but Not the Exclusive, Cause of Ectopic Mineralization in an Abcc6–/– Mouse Model of PXE

Pseudoxanthoma elasticum (PXE), a prototype of heritable ectopic mineralization disorders, is caused in most cases by inactivating mutations in the ABCC6 gene. It was recently discovered that absence of ABCC6-mediated adenosine triphosphate release from the liver and consequently reduced plasma inorganic pyrophosphate (PPi) levels underlie PXE. This study examined whether reduced levels of circulating PPi, an antimineralization factor, is the sole mechanism of PXE. The Abcc6-/- and Enpp1asj mice were crossed with transgenic mice expressing human ENPP1, an ectonucleotidase that generates PPi from adenosine triphosphate. We generated Abcc6-/- and Enpp1asj mice, either wild-type or hemizygous for human ENPP1. Plasma levels of PPi and the degree of ectopic mineralization were determined. Overexpression of human ENPP1 in Enpp1asj mice normalized plasma PPi levels to that of wild-type mice and, consequently, completely prevented ectopic mineralization. These changes were accompanied by restoration of their bone microarchitecture. In contrast, although significantly reduced mineralization was noted in Abcc6-/- mice expressing human ENPP1, small mineralization foci were still evident despite increased plasma PPi levels. These results suggest that PPi is the major mediator of ectopic mineralization in PXE, but there might be an alternative, as yet unknown mechanism, independent of PPi, by which ABCC6 prevents ectopic mineralization under physiologic conditions.

501 Plasma PPi is the major, but not exclusive, cause of ectopic mineralization in pseudoxanthoma elasticum

Pseudoxanthoma elasticum (PXE), a prototype of heritable ectopic mineralization disorders, affects the skin, eyes and the arterial blood vessels. PXE is caused by inactivating mutations in the ABCC6 gene and the disease is characterized by calcium hydroxyapatite deposition on connective tissues. It was recently discovered that absence of ABCC6-mediated ATP release from the liver and consequently reduced plasma PPi levels underlie PXE. In this study, we examined whether elevated circulating levels of PPi, a powerful mineralization inhibitor, is the sole mechanism by which ABCC6 counteracts ectopic mineralization in PXE. Abcc6-/- mice, a model of PXE, were crossed with transgenic mice with ubiquitous expression of human ENPP1, an ectonucleotidase which generates PPi from ATP. We generated Abcc6-/-mice either wild type or hemizygous for human ENPP1. The Enpp1asj mice, a model for generalized arterial calcification of infancy caused by ENPP1 mutations, were also crossed with human ENPP1 transgenic mice to compensate for the loss of the endogenous mouse protein. Plasma levels of PPi and the degree of mineralization of connective tissue capsule of vibrissae, a reliable biomarker of the mineralization process in these mice, were determined. Overexpression of human ENPP1 in Enpp1asj mice normalized plasma PPi levels to that of wild type mice, and consequently, completely prevented mineralization. In contrast, significantly reduced mineralization was noted in Abcc6-/- mice with overexpression of human ENPP1, however, with small mineralization foci still evident despite increased plasma PPi levels. These results suggest that PPi is the major mediator of connective tissue mineralization in PXE, but there is a second, as-yet unknown, alternative mechanism other than PPi by which ABCC6 prevents ectopic mineralization under physiologic conditions.

Insights into Pathomechanisms and Treatment Development in Heritable Ectopic Mineralization Disorders: Summary of the PXE International Biennial Research Symposium—2016

Pseudoxanthoma elasticum is a prototype of heritable ectopic mineralization disorders, with phenotypic overlap with generalized arterial calcification of infancy and arterial calcification due to CD73 deficiency. Recent observations have suggested that the reduced inorganic pyrophosphate/phosphate ratio is the cause of soft connective tissue mineralization in these disorders. PXE International, a patient advocacy organization, supports research in part by sponsoring biennial research symposia on these disorders; the latest meeting was held in September 2016 at Thomas Jefferson University, Philadelphia. This report summarizes the progress in pseudoxanthoma elasticum and other ectopic mineralization disorders, as presented in the symposium, with focus on translational aspects of precision medicine toward improved diagnostics and treatment development for these currently intractable disorders.

Abcc6 Knockout Rat Model Highlights the Role of Liver in PPi Homeostasis in Pseudoxanthoma Elasticum

Pseudoxanthoma elasticum, a heritable ectopic mineralization disorder, is caused by mutations in the ABCC6 gene primarily expressed in the liver and the kidneys. The fundamental question on pathogenesis of pseudoxanthoma elasticum, whether lack of ABCC6 expression in liver or kidney is the primary site of molecular pathology in peripheral tissues, has not been addressed. We generated a series of Abcc6-/- rats as models of pseudoxanthoma elasticum depicting ectopic mineralization in the skin, eyes, and the arterial blood vessels. Plasma inorganic pyrophosphate (PPi) level was reduced (<30%) in the Abcc6-/- rats leading to a lowered PPi/inorganic phosphate plasma ratio. In situ liver and kidney perfusions were performed to determine the relative contribution of these organs to PPi levels in circulation. PPi levels in the perfusates both in the liver and kidney of Abcc6-/- rats were significantly reduced, but the PPi levels in the liver perfusates of wild-type rats were 10-fold higher than that in the kidney perfusates. These observations suggest a critical role of hepatic ABCC6 in contributing to plasma PPi levels, identifying liver as a target of molecular correction to counteract ectopic mineralization in pseudoxanthoma elasticum.

Research Progress in Pseudoxanthoma Elasticum and Related Ectopic Mineralization Disorders

Heritable ectopic mineralization disorders represent a phenotypically diverse group of conditions characterized by deposition of calcium phosphate complexes in soft connective tissues. The prototype of such conditions is pseudoxanthoma elasticum (PXE), and related conditions with overlapping clinical features include generalized arterial calcification of infancy (GACI) and arterial calcification due to CD73 deficiency (ACDC). Molecular genetic investigations have revealed mutations in the genes physiologically involved in generation of inorganic pyrophosphate (PPi) and phosphate (Pi), and the findings suggest a unifying pathomechanism relating to reduced PPi/Pi ratio. This hypothesis is based on the notion that PPi serves as a powerful inhibitor of mineralization while Pi is a pro-mineralization factor, and an appropriate PPi/Pi ratio is critical for prevention of ectopic mineralization under homeostatic conditions.PXE International, the premiere patient support organization, advocating on behalf of patients and families with PXE, sponsors regular research meetings evaluating the progress in this and related ectopic mineralization disorders. The latest meetings were held in September 2014 in Bethesda, MD and in September 2015 in Budapest, Hungary. This report summarizes the latest progress in research on PXE and related ectopic mineralization disorders, based on presentations and discussions in these meetings, with pharmacologic implications for currently intractable disorders.

Calcinosis: pathophysiology and management.

This article reviews the most updated literature regarding the epidemiology, pathophysiology, diagnosis, and treatment of calcinosis cutis in patients with systemic sclerosis (SSc). Our review identified observational studies that describe the frequency of calcinosis in SSc and associated clinical features, genetic studies in animal models of heritable disorders leading to calcium deposition, and case series and case reports describing new diagnostic approaches and therapeutic interventions. Calcinosis cutis is the deposition of calcium in the skin and subcutaneous tissues. It affects almost one quarter of patients with SSc, and is associated with longer disease duration, digital ulcers, acroosteolysis, positive anticentromere antibody, and positive anti-PM/Scl antibody. Local trauma, chronic inflammation, and vascular hypoxia have been proposed as potential pathomechanisms. The development of mouse models that mimic heritable ectopic mineralization disorders are contributing to the understanding of the process of calcification. Diagnosis can be made clinically or with plain radiography. Experimental diagnostic studies include ultrasonography, multidetector computed tomography, and dual-energy computed tomography. Several pharmacologic therapies have been tried for calcinosis with variable results, but surgical excision of calcium deposits remains the mainstay of treatment.