Genetics in arterial calcification: pieces of a puzzle and cogs in a wheel.
ABSTRACT: Artery calcification reflects an admixture of factors such as ectopic osteochondral differentiation with primary host pathological conditions. We review how genetic factors, as identified by human genome-wide association studies, and incomplete correlations with various mouse studies, including knockout and strain analyses, fit into "pieces of the puzzle" in intimal calcification in human atherosclerosis, and artery tunica media calcification in aging, diabetes mellitus, and chronic kidney disease. We also describe in sharp contrast how ENPP1, CD73, and ABCC6 serve as "cogs in a wheel" of arterial calcification. Specifically, each is a minor component in the function of a much larger network of factors that exert balanced effects to promote and suppress arterial calcification. For the network to normally suppress spontaneous arterial calcification, the "cogs" ENPP1, CD73, and ABCC6 must be present and in working order. Monogenic ENPP1, CD73, and ABCC6 deficiencies each drive a molecular pathophysiology of closely related but phenotypically different diseases (generalized arterial calcification of infancy (GACI), pseudoxanthoma elasticum (PXE) and arterial calcification caused by CD73 deficiency (ACDC)), in which premature onset arterial calcification is a prominent but not the sole feature.
Project description: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 <i>ABCC6</i>, 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 <i>ENPP1</i> and <i>NT5E</i>, 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.
Project description:Ectopic mineralization is a global problem and leading cause of morbidity and mortality. The pathomechanisms of ectopic mineralization are poorly understood. Recent studies on heritable ectopic mineralization disorders with defined gene defects have been helpful in elucidation of the mechanisms of ectopic mineralization in general. The prototype of such disorders is pseudoxanthoma elasticum (PXE), a late-onset, slowly progressing disorder with multisystem clinical manifestations. Other conditions include generalized arterial calcification of infancy (GACI), characterized by severe, early-onset mineralization of the cardiovascular system, often with early postnatal demise. In addition, arterial calcification due to CD73 deficiency (ACDC) occurs late in life, mostly affecting arteries in the lower extremities in elderly individuals. These three conditions, PXE, GACI, and ACDC, caused by mutations in ABCC6, ENPP1, and NT5E, respectively, are characterized by reduced levels of inorganic pyrophosphate (PPi) in plasma. Because PPi is a powerful antimineralization factor, it has been postulated that reduced PPi is a major determinant for ectopic mineralization in these conditions. These and related observations on complementary mechanisms of ectopic mineralization have resulted in development of potential treatment modalities for PXE, including administration of bisphosphonates, stable PPi analogs with antimineralization activity. It is conceivable that efficient treatments may soon become available for heritable ectopic mineralization disorders with application to common calcification disorders.
Project description:Generalized arterial calcification of infancy (GACI) is associated with biallelic mutations in ENPP1 in the majority of cases, whereas mutations in ABCC6 (ATP-binding cassette subfamily C number 6) are known to cause pseudoxanthoma elasticum (PXE). However, ABCC6 mutations account for a significant subset of GACI cases, and ENPP1 mutations can also be associated with PXE lesions. Based on the considerable overlap of GACI and PXE, both entities appear to reflect two ends of a clinical spectrum of ectopic calcification rather than two distinct disorders. ABCC6 and ENPP1 mutations might lead to alterations of the same physiological pathways.
Project description:Spontaneous pathologic arterial calcifications in childhood can occur in generalized arterial calcification of infancy (GACI) or in pseudoxanthoma elasticum (PXE). GACI is associated with biallelic mutations in ENPP1 in the majority of cases, whereas mutations in ABCC6 are known to cause PXE. However, the genetic basis in subsets of both disease phenotypes remains elusive. We hypothesized that GACI and PXE are in a closely related spectrum of disease. We used a standardized questionnaire to retrospectively evaluate the phenotype of 92 probands with a clinical history of GACI. We obtained the ENPP1 genotype by conventional sequencing. In those patients with less than two disease-causing ENPP1 mutations, we sequenced ABCC6. We observed that three GACI patients who carried biallelic ENPP1 mutations developed typical signs of PXE between 5 and 8 years of age; these signs included angioid streaks and pseudoxanthomatous skin lesions. In 28 patients, no disease-causing ENPP1 mutation was found. In 14 of these patients, we detected pathogenic ABCC6 mutations (biallelic mutations in eight patients, monoallelic mutations in six patients). Thus, ABCC6 mutations account for a significant subset of GACI patients, and ENPP1 mutations can also be associated with PXE lesions in school-aged children. Based on the considerable overlap of genotype and phenotype of GACI and PXE, both entities appear to reflect two ends of a clinical spectrum of ectopic calcification and other organ pathologies, rather than two distinct disorders. ABCC6 and ENPP1 mutations might lead to alterations of the same physiological pathways in tissues beyond the artery.
Project description:Generalized arterial calcification of infancy (GACI) is an autosomal recessive disorder characterized by congenital calcification of large- and medium-sized arteries, associated with early myocardial infarction, heart failure, and stroke, and premature death. Most cases of GACI are caused by mutations in the ENPP1 gene. We first studied two siblings with GACI from a non-consanguineous family without mutations in the ENPP1 gene. To search for disease-causing mutations, we identified genomic regions shared between the two affected siblings but not their unaffected parents or brother. The ABCC6 gene, which is mutated in pseudoxanthoma elasticum (PXE), resided within a small region of homozygosity shared by the affected siblings. Sequence analysis of ABCC6 revealed that the two affected siblings were homozygous for the missense mutation p.R1314W. Subsequently, ABCC6 mutations were identified in five additional GACI families with normal ENPP1 sequences. Genetic mutations in ABCC6 in patients with PXE are associated with ectopic tissue mineralization in the skin and arterial blood vessels. Thus, our findings provide additional evidence that the ABCC6 gene product inhibits calcification under physiologic conditions and confirm a second locus for GACI. In addition, our study emphasizes the potential utility of shared homozygosity mapping to identify genetic causes of inherited disorders.
Project description:Arterial Calcification due to Deficiency of CD73 (ACDC) results from mutations in the NT5E gene encoding the 5' exonucleotidase, CD73. We now describe the third familial case of ACDC, including radiological and histopathological details of the arterial calcifications. The medial lesions involve the entire circumference of the elastic lamina, in contrast to the intimal plaque-like disease of atherosclerosis. The demonstration of broken and fragmented elastic fibers leading to generalized vascular calcification suggests an analogy to pseudoxanthoma elasticum (PXE), which exhibits similar histopathology. Classical PXE is caused by deficiency of ABCC6, a C type ABC transporter whose ligand is unknown. Other C type ABC proteins transport nucleotides, so the newly described role of adenosine in inhibiting vascular calcification, along with the similarity of ACDC and PXE with respect to vascular pathology, suggests that adenosine may be the ligand for ABCC6.
Project description:<h4>Purpose</h4>Generalized arterial calcification of infancy (GACI), characterized by vascular calcifications that are often fatal shortly after birth, is usually caused by deficiency of ENPP1. A small fraction of GACI cases result from deficiency of ABCC6, a membrane transporter. The natural history of GACI survivors has not been established in a prospective fashion.<h4>Methods</h4>We performed deep phenotyping of 20 GACI survivors.<h4>Results</h4>Sixteen of 20 subjects presented with arterial calcifications, but only 5 had residual involvement at the time of evaluation. Individuals with ENPP1 deficiency either had hypophosphatemic rickets or were predicted to develop it by 14 years of age; 14/16 had elevated intact FGF23 levels (iFGF23). Blood phosphate levels correlated inversely with iFGF23. For ENPP1-deficient individuals, the lifetime risk of cervical spine fusion was 25%, that of hearing loss was 75%, and the main morbidity in adults was related to enthesis calcification. Four ENPP1-deficient individuals manifested classic skin or retinal findings of PXE. We estimated the minimal incidence of ENPP1 deficiency at ~1 in 200,000 pregnancies.<h4>Conclusion</h4>GACI appears to be more common than previously thought, with an expanding spectrum of overlapping phenotypes. The relationships among decreased ENPP1, increased iFGF23, and rickets could inform future therapies.
Project description:Various disorders including pseudoxanthoma elasticum (PXE) and generalized arterial calcification of infancy (GACI), which are caused by inactivating mutations in ABCC6 and ENPP1, respectively, present with extensive tissue calcification due to reduced plasma pyrophosphate (PPi). However, it has always been assumed that the bioavailability of orally administered PPi is negligible. Here, we demonstrate increased PPi concentration in the circulation of humans after oral PPi administration. Furthermore, in mouse models of PXE and GACI, oral PPi provided via drinking water attenuated their ectopic calcification phenotype. Noticeably, provision of drinking water with 0.3 mM PPi to mice heterozygous for inactivating mutations in Enpp1 during pregnancy robustly inhibited ectopic calcification in their Enpp1-/- offspring. Our work shows that orally administered PPi is readily absorbed in humans and mice and inhibits connective tissue calcification in mouse models of PXE and GACI PPi, which is recognized as safe by the FDA, therefore not only has great potential as an effective and extremely low-cost treatment for these currently intractable genetic disorders, but also in other conditions involving connective tissue calcification.
Project description:Pathological (ectopic) mineralization of soft tissues occurs during aging, in several common conditions such as diabetes, hypercholesterolemia, and renal failure and in certain genetic disorders. Pseudoxanthoma elasticum (PXE), a multi-organ disease affecting dermal, ocular, and cardiovascular tissues, is a model for ectopic mineralization disorders. ABCC6 dysfunction is the primary cause of PXE, but also some cases of generalized arterial calcification of infancy (GACI). ABCC6 deficiency in mice underlies an inducible dystrophic cardiac calcification phenotype (DCC). These calcification diseases are part of a spectrum of mineralization disorders that also includes Calcification of Joints and Arteries (CALJA). Since the identification of ABCC6 as the "PXE gene" and the development of several animal models (mice, rat, and zebrafish), there has been significant progress in our understanding of the molecular genetics, the clinical phenotypes, and pathogenesis of these diseases, which share similarities with more common conditions with abnormal calcification. ABCC6 facilitates the cellular efflux of ATP, which is rapidly converted into inorganic pyrophosphate (PPi) and adenosine by the ectonucleotidases NPP1 and CD73 (NT5E). PPi is a potent endogenous inhibitor of calcification, whereas adenosine indirectly contributes to calcification inhibition by suppressing the synthesis of tissue non-specific alkaline phosphatase (TNAP). At present, therapies only exist to alleviate symptoms for both PXE and GACI; however, extensive studies have resulted in several novel approaches to treating PXE and GACI. This review seeks to summarize the role of ABCC6 in ectopic calcification in PXE and other calcification disorders, and discuss therapeutic strategies targeting various proteins in the pathway (ABCC6, NPP1, and TNAP) and direct inhibition of calcification via supplementation by various compounds.
Project description:Generalized arterial calcification of infancy (GACI) is associated with widespread arterial calcification and stenoses and is caused by mutations in ENPP1. ENPP1 encodes for ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which cleaves ATP to generate inorganic pyrophosphate (PPi) and adenosine monophosphate (AMP) extracellularly. The current study was designed to define the prevalence of arterial stenoses in GACI individuals and to identify the mechanism through which ENPP1 deficiency causes intimal proliferation. Furthermore, we aimed to effectively prevent and treat neointima formation in an animal model of GACI through the systemic administration of recombinant human (rh)ENPP1-Fc protein. Based on a literature review, we report that arterial stenoses are present in at least 72.4% of GACI cases. We evaluated the effect of rhENPP1-Fc on ENPP1-silenced human vascular smooth muscle cells (VSMCs) and on induced intimal proliferation in Enpp1-deficient ttw/ttw mice treated with carotid ligation. We demonstrate that silencing ENPP1 in VSMCs resulted in a tenfold increase in proliferation relative to that of cells transfected with negative control siRNA. The addition of rhENPP1-Fc, AMP or adenosine restored the silenced ENPP1-associated proliferation. In contrast, neither PPi nor etidronate, a current off-label treatment for GACI, had an effect on VSMC proliferation. Furthermore, subcutaneous rhENPP1-Fc protein replacement was effective in preventing and treating intimal hyperplasia induced by carotid ligation in an animal model of GACI. We conclude that ENPP1 inhibits neointima formation by generating AMP. RhENPP1-Fc may serve as an approach for the effective prevention and treatment of arterial stenoses in GACI.