RESUMEN

In previous studies, we have demonstrated that stress response-induced high glucocorticoid levels could be the underlying cause of traumatic heterotopic ossification (HO), and we have developed a glucocorticoid-induced ectopic mineralization (EM) mouse model by systemic administration of a high dose of dexamethasone (DEX) to animals with muscle injury induced by cardiotoxin injection. In this model, dystrophic calcification (DC) developed into HO in a cell autonomous manner. However, it is not clear how DC is formed after DEX treatment. Therefore, in this study, we aimed to explore how glucocorticoids initiate muscle EM at a cellular and molecular level. We showed that DEX treatment inhibited inflammatory cell infiltration into injured muscle but inflammatory cytokine production in the muscle was significantly increased, suggesting that other non-inflammatory muscle cell types may regulate the inflammatory response and the muscle repair process. Accompanying this phenotype, transforming growth factor ß1 (TGF-ß1) expression in fibro-adipogenic progenitors (FAPs) was greatly downregulated. Since TGF-ß1 is a strong immune suppressor and FAP's regulatory role has a large impact on muscle repair, we hypothesized that downregulation of TGF-ß1 in FAPs after DEX treatment resulted in this hyperinflammatory state and subsequent failed muscle repair and EM formation. To test our hypothesis, we utilized a transgenic mouse model to specifically knockout Tgfb1 gene in PDGFRα-positive FAPs to investigate if the transgenic mice could recapitulate the phenotype that was induced by DEX treatment. Our results showed that the transgenic mice completely phenocopied this hyperinflammatory state and spontaneously developed EM following muscle injury. On the contrary, therapeutics that enhanced TGF-ß1 signaling in FAPs inhibited the inflammatory response and attenuated muscle EM. In summary, these results indicate that FAPs-derived TGF-ß1 is a key molecule in regulating muscle inflammatory response and subsequent EM, and that glucocorticoids exert their effect via downregulating TGF-ß1 in FAPs.

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Heterotopic ossification (HO) is abnormal bone formation in soft tissue. Glucocorticoids, which have strong anti-inflammatory properties, have usually been used as HO therapeutics. However, our findings suggest that glucocorticoids can also promote HO formation. In this study, we tried to explain the underlying reason for these seemingly contradictory observations. We showed that glucocorticoids, in addition to exerting an anti-inflammatory effect on inflammatory cells, can also target another type of muscle cell to exert a proinflammatory effect. These cells are called fibro-adipogenic progenitors (FAPs), and we demonstrated that FAPs played a master regulatory role in the muscle inflammatory response by modulating the expression of transforming growth factor ß1 (TGF-ß1), a well-known immune suppressor. In summary, our findings highlighted the importance of FAP TGF-ß1 levels in affecting the progression and regression of muscle HO and provided new treatment options for HO based on their ability to elevate TGF-ß1 levels in FAPs.

Asunto(s)

Dexametasona , Regulación hacia Abajo , Células Madre , Factor de Crecimiento Transformador beta1 , Animales , Factor de Crecimiento Transformador beta1/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Ratones , Dexametasona/farmacología , Células Madre/metabolismo , Células Madre/efectos de los fármacos , Adipogénesis/efectos de los fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Músculo Esquelético/efectos de los fármacos

RESUMEN

Raine syndrome (RNS) is a rare autosomal recessive osteosclerotic dysplasia. RNS is caused by loss-of-function disease-causative variants of the FAM20C gene that encodes a kinase that phosphorylates most of the secreted proteins found in the body fluids and extracellular matrix. The most common RNS clinical features are generalized osteosclerosis, facial dysmorphism, intracerebral calcifications and respiratory defects. In non-lethal RNS forms, oral traits include a well-studied hypoplastic amelogenesis imperfecta (AI) and a much less characterized gingival phenotype. We used immunomorphological, biochemical, and siRNA approaches to analyze gingival tissues and primary cultures of gingival fibroblasts of two unrelated, previously reported RNS patients. We showed that fibrosis, pathological gingival calcifications and increased expression of various profibrotic and pro-osteogenic proteins such as POSTN, SPARC and VIM were common findings. Proteomic analysis of differentially expressed proteins demonstrated that proteins involved in extracellular matrix (ECM) regulation and related to the TGFß/SMAD signaling pathway were increased. Functional analyses confirmed the upregulation of TGFß/SMAD signaling and subsequently uncovered the involvement of two closely related transcription cofactors important in fibrogenesis, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). Knocking down of FAM20C confirmed the TGFß-YAP/TAZ interplay indicating that a profibrotic loop enabled gingival fibrosis in RNS patients. In summary, our in vivo and in vitro data provide a detailed description of the RNS gingival phenotype. They show that gingival fibrosis and calcifications are associated with, and most likely caused by excessed ECM production and disorganization. They furthermore uncover the contribution of increased TGFß-YAP/TAZ signaling in the pathogenesis of the gingival fibrosis.

Asunto(s)

Anomalías Múltiples , Proteínas Adaptadoras Transductoras de Señales , Fisura del Paladar , Hipoplasia del Esmalte Dental , Exoftalmia , Fibroblastos , Fibrosis , Encía , Osteosclerosis , Proteómica , Transducción de Señal , Factores de Transcripción , Factor de Crecimiento Transformador beta , Proteínas Señalizadoras YAP , Humanos , Factor de Crecimiento Transformador beta/metabolismo , Encía/metabolismo , Encía/patología , Proteómica/métodos , Fibrosis/metabolismo , Proteínas Señalizadoras YAP/metabolismo , Proteínas Señalizadoras YAP/genética , Osteosclerosis/metabolismo , Osteosclerosis/genética , Osteosclerosis/patología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Hipoplasia del Esmalte Dental/metabolismo , Hipoplasia del Esmalte Dental/genética , Hipoplasia del Esmalte Dental/patología , Fibroblastos/metabolismo , Fibroblastos/patología , Microcefalia/metabolismo , Microcefalia/genética , Microcefalia/patología , Femenino , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ/metabolismo , Masculino , Transactivadores/metabolismo , Transactivadores/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Quinasa de la Caseína I/metabolismo , Quinasa de la Caseína I/genética , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/genética , Amelogénesis Imperfecta/metabolismo , Amelogénesis Imperfecta/genética , Amelogénesis Imperfecta/patología , Células Cultivadas

RESUMEN

Generalized arterial calcification of infancy (GACI) is a rare genetic disease characterized by arterial calcifications or stenoses and hypertension. GACI is caused by mutations in the ENPP1 or ABCC6 genes, and it often causes intrauterine or early infancy death. Here, we report a case of rare GACI caused by a homozygous variation in ENPP1, in a Chinese infant initially presenting with hypertension. The proband was an 8-month-old boy with in utero tricuspid valve calcification, presenting with hypertension at birth. Enhanced computed tomography revealed extensive arterial calcification. Genetic testing identified a homozygous variation in ENPP1 (c.783C > G p.Y261X), which led to the diagnosis of GACI. This mutation has been reported in only three Chinese patients, which all initially presented with hypophosphatemic rickets rather than GACI. This case enriches the clinical and genetic spectrum of ENPP1 mutations and reminds us that GACI should be considered in an infant presenting with hypertension and extensive arterial calcification, and that genetic testing should be performed.

RESUMEN

BACKGROUND: Pseudoxanthoma elasticum (PXE), a monogenic disorder resulting in calcification affecting the skin, eyes and peripheral arteries, is caused by mutations in the ABCC6 gene, and is associated with low plasma inorganic pyrophosphate (PPi). It is unknown how ABCC6 genotype affects plasma PPi. METHODS: We studied the association of ABCC6 genotype (192 patients with biallelic pathogenic ABCC6 mutations) and PPi levels, and its association with the severity of arterial and ophthalmological phenotypes. ABCC6 variants were classified as truncating or non-truncating, and three groups of the 192 patients were formed: those with truncating mutations on both chromosomes (n = 121), those with two non-truncating mutations (n = 10), and a group who had one truncating and one non-truncating ABCC6 mutation (n = 61). The hypothesis formulated before this study was that there was a negative association between PPi level and disease severity. RESULTS: Our findings confirm low PPi in PXE compared with healthy controls (0.53 ± 0.15 vs. 1.13 ± 0.29 µM, p < 0.01). The PPi of patients correlated with increasing age (ß: 0.05 µM, 95% CI: 0.03-0.06 per 10 years) and was higher in females (0.55 ± 0.17 vs. 0.51 ± 0.13 µM in males, p = 0.03). However, no association between PPi and PXE phenotypes was found. When adjusted for age and sex, no association between PPi and ABCC6 genotype was found. CONCLUSIONS: Our data suggest that the relationship between ABCC6 mutations and reduced plasma PPi may not be as direct as previously thought. PPi levels varied widely, even in patients with the same ABCC6 mutations, further suggesting a lack of direct correlation between them, even though the ABCC6 protein-mediated pathway is responsible for ~60% of this metabolite in the circulation. We discuss potential factors that may perturb the expected associations between ABCC6 genotype and PPi and between PPi and disease severity. Our findings support the argument that predictions of pathogenicity made on the basis of mutations (or on the structure of the mutated protein) could be misleading.

RESUMEN

Inorganic pyrophosphate (PPi) is a crucial extracellular mineralization regulator. Low plasma PPi concentrations underlie the soft tissue calcification present in several rare hereditary mineralization disorders as well as in more common conditions like chronic kidney disease and diabetes. Even though deregulated plasma PPi homeostasis is known to be linked to multiple human diseases, there is currently no reliable assay for its quantification. We here describe a PPi assay that employs the enzyme ATP sulfurylase to convert PPi into ATP. Generated ATP is subsequently quantified by firefly luciferase-based bioluminescence. An internal ATP standard was used to correct for sample-specific interference by matrix compounds on firefly luciferase activity. The assay was validated and shows excellent precision (< 3.5%) and accuracy (93-106%) of PPi spiked into human plasma samples. We found that of several anticoagulants tested only EDTA effectively blocked conversion of ATP into PPi in plasma after blood collection. Moreover, filtration over a 300,000-Da molecular weight cut-off membrane reduced variability of plasma PPi and removed ATP present in a membrane-enclosed compartment, possibly platelets. Applied to plasma samples of wild-type and Abcc6-/- rats, an animal model with established low circulating levels of PPi, the new assay showed lower variability than the assay that was previously in routine use in our laboratory. In conclusion, we here report a new and robust assay to determine PPi concentrations in plasma, which outperforms currently available assays because of its high sensitivity, precision, and accuracy.

Asunto(s)

Calcinosis , Difosfatos , Humanos , Ratas , Animales , Luciferasas de Luciérnaga , Adenosina Trifosfato

RESUMEN

Ectopic mineralization refers to the deposition of mineralized complexes in the extracellular matrix of soft tissues. Calcific aortic valve disease, vascular calcification, gallstones, kidney stones, and abnormal mineralization in arthritis are common examples of ectopic mineralization. They are debilitating diseases and exhibit excess mortality, disability, and morbidity, which impose on patients with limited social or financial resources. Recent recognition that inflammation plays an important role in ectopic mineralization has attracted the attention of scientists from different research fields. In the present review, we summarize the origin of inflammation in ectopic mineralization and different channels whereby inflammation drives the initiation and progression of ectopic mineralization. The current knowledge of inflammatory milieu in pathological mineralization is reviewed, including how immune cells, pro-inflammatory mediators, and osteogenic signaling pathways induce the osteogenic transition of connective tissue cells, providing nucleating sites and assembly of aberrant minerals. Advances in the understanding of the underlying mechanisms involved in inflammatory-mediated ectopic mineralization enable novel strategies to be developed that may lead to the resolution of these enervating conditions.

Asunto(s)

Calcio , Hormona Paratiroidea

RESUMEN

Pseudoxanthoma elasticum (PXE, OMIM # 264800) is an autosomal recessive, multisystemic disorder, associated with mutations of the ABCC6 gene. Ectopic mineralization is in the background of the clinical manifestations of the disease. Calcium-salt crystals are deposited primarily in the skin, in the Bruch membrane of the eyes, and in the vascular endothelium. Thus, in addition to the skin lesions, visual impairment and cardiovascular involvement also occur. Clinical symptoms show varying severity and display heterogeneous appearance. The identification of the phenotype and care of the patients require a multidisciplinary perspective based on the collaboration of a dermatologist, ophthalmologist, cardiologist, and clinical geneticist. The aim of our work is to describe the development of symptoms of the disease, in order to facilitate the diagnosis. In addition, we aim to draw attention to the importance of early diagnosis of pseudoxanthoma elasticum, and to present modern diagnostic methods. Considering the development of severe systemic complications, the early diagnosis with the collaboration between related specialists is crucial to provide optimal clinical care and management of the patients.

Asunto(s)

Seudoxantoma Elástico , Lámina Basal de la Coroides , Humanos , Mutación , Fenotipo , Seudoxantoma Elástico/diagnóstico , Seudoxantoma Elástico/genética , Piel/patología

RESUMEN

Characteristic skin changes lead to diagnosis of pseudoxanthoma elasticum (PXE), an ectopic mineralization disorder, involving primarily the skin, eyesight, and arterial vessels. Early recognition is crucial for timely treatment of extracutaneous complications. We hereby report a series of four cases of PXE with pathognomonic skin lesions and a broad spectrum of systemic complications.

Asunto(s)

Seudoxantoma Elástico , Dermatólogos , Humanos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Seudoxantoma Elástico/diagnóstico , Piel/patología

RESUMEN

Chronic low back pain is a highly prevalent health condition intricately linked to intervertebral disc degeneration. One of the prominent features of disc degeneration that is commonly observed with aging is dystrophic calcification. ATP-binding cassette sub-family C member 6 (ABCC6), a presumed ATP efflux transporter, is a key regulator of systemic levels of the mineralization inhibitor pyrophosphate (PPi). Mutations in ABCC6 result in pseudoxanthoma elasticum (PXE), a progressive human metabolic disorder characterized by mineralization of the skin and elastic tissues. The implications of ABCC6 loss-of-function on pathological mineralization of structures in the spine, however, are unknown. Using the Abcc6 -/- mouse model of PXE, we investigated age-dependent changes in the vertebral bone and intervertebral disc. Abcc6 -/- mice exhibited diminished trabecular bone quality parameters at 7 months, which remained significantly lower than the wild-type mice at 18 months of age. Abcc6 -/- vertebrae showed increased TRAP staining along with decreased TNAP staining, suggesting an enhanced bone resorption as well as decreased bone formation. Surprisingly, however, loss of ABCC6 resulted only in a mild, aging disc phenotype without evidence of dystrophic mineralization. Finally, we tested the utility of oral K3Citrate to treat the vertebral phenotype since it is shown to regulate hydroxyapatite mechanical behavior. The treatment resulted in inhibition of the osteoclastic response and an early improvement in mechanical properties of the bone underscoring the promise of potassium citrate as a therapeutic agent. Our data suggest that although ectopic mineralization is tightly regulated in the disc, loss of ABCC6 compromises vertebral bone quality and dysregulates osteoblast-osteoclast coupling.

RESUMEN

Dystrophic calcification is a common histopathologic finding that can be concomitant with a plethora of diseases, ranging from self-limited infections to insidious malignancies. Gamna-Gandy bodies (GGBs) are a form of dystrophic calcification associated with chronic hemolysis and are typically observed in the spleen. In this report, we present the case of a 92-year-old man who presented with a 4-mm blue papule that was biopsied given the concern for a blue nevus. The subsequent histopathologic examination of the biopsy specimen showed a dermal organizing hematoma adjacent to pale-yellow to brown, refractile material within fibrotic collagen consistent with GGBs. Scanning electron microscopy with energy-dispersive x-ray analysis (SEM/EDXA) revealed that the structures were composed of carbon (39%), oxygen (32%), iron (16%), phosphorus (7%), calcium (5%), and sodium (1%). Fourier transform infrared spectroscopy identified amorphous calcium phosphate. GGBs have not been previously described in the skin and have been rarely characterized with SEM/EDXA in other sites.

Asunto(s)

Calcinosis , Bazo , Anciano de 80 o más Años , Calcinosis/patología , Calcio , Humanos , Masculino , Bazo/patología

RESUMEN

PURPOSE: Heritable 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. METHODS: Sequence 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. RESULTS: A 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. CONCLUSION: The 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.

Asunto(s)

Heterogeneidad Genética , Seudoxantoma Elástico , Estudios de Cohortes , Tejido Conectivo/patología , Humanos , Mutación Missense , Seudoxantoma Elástico/genética

RESUMEN

OBJECTIVE: The pathogenesis of calcinosis cutis, a disabling complication of SSc, is poorly understood and effective treatments are lacking. Inorganic pyrophosphate (PPi) is a key regulator of ectopic mineralization, and its deficiency has been implicated in ectopic mineralization disorders. We therefore sought to test the hypothesis that SSc may be associated with reduced circulating PPi, which might play a pathogenic role in calcinosis cutis. METHODS: Subjects with SSc and age-matched controls without SSc were recruited from the outpatient rheumatology clinics at Rutgers and Northwestern Universities (US cohort), and from the Universities of Szeged and Debrecen (Hungarian cohort). Calcinosis cutis was confirmed by direct palpation, by imaging or both. Plasma PPi levels were determined in platelet-free plasma using ATP sulfurylase to convert PPi into ATP in the presence of excess adenosine 5' phosphosulfate. RESULTS: Eighty-one patients with SSc (40 diffuse cutaneous, and 41 limited cutaneous SSc) in the US cohort and 45 patients with SSc (19 diffuse cutaneous and 26 limited cutaneous SSc) in the Hungarian cohort were enrolled. Calcinosis was frequently detected (40% of US and 46% of the Hungarian cohort). Plasma PPi levels were significantly reduced in both SSc cohorts with and without calcinosis (US: P = 0.003; Hungarian: P < 0.001). CONCLUSIONS: Circulating PPi are significantly reduced in SSc patients with or without calcinosis. Reduced PPi may be important in the pathophysiology of calcinosis and contribute to tissue damage with chronic SSc. Administering PPi may be a therapeutic strategy and larger clinical studies are planned to confirm our findings.

Asunto(s)

Calcinosis/sangre , Calcinosis/etiología , Difosfatos/sangre , Esclerodermia Sistémica/sangre , Esclerodermia Sistémica/complicaciones , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad

RESUMEN

Pseudoxanthoma elasticum (PXE; OMIM 264800) is a rare heritable multisystem disorder, characterized by ectopic mineralization affecting elastic fibres in the skin, eyes and the cardiovascular system. Skin findings often lead to early diagnosis of PXE, but currently, no specific treatment exists to counteract the progression of symptoms. PXE belongs to a group of Mendelian calcification disorders linked to pyrophosphate metabolism, which also includes generalized arterial calcification of infancy (GACI) and arterial calcification due to CD73 deficiency (ACDC). Inactivating mutations in ABCC6, ENPP1 and NT5E are the genetic cause of these diseases, respectively, and all of them result in reduced inorganic pyrophosphate (PPi ) concentration in the circulation. Although PPi is a strong inhibitor of ectopic calcification, oral supplementation therapy was initially not considered because of its low bioavailability. Our earlier work however demonstrated that orally administered pyrophosphate inhibits ectopic calcification in the animal models of PXE and GACI, and that orally given Na4 P2 O7 is absorbed in humans. Here, we report that gelatin-encapsulated Na2 H2 P2 O7  has similar absorption properties in healthy volunteers and people affected by PXE. The sodium-free K2 H2 P2 O7 form resulted in similar uptake in healthy volunteers and inhibited calcification in Abcc6-/- mice as effectively as its sodium counterpart. Novel pyrophosphate compounds showing higher bioavailability in mice were also identified. Our results provide an important step towards testing oral PPi in clinical trials in PXE, or potentially any condition accompanied by ectopic calcification including diabetes, chronic kidney disease or ageing.

Asunto(s)

Seudoxantoma Elástico , Calcificación Vascular , Animales , Suplementos Dietéticos , Difosfatos , Humanos , Ratones , Mutación , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/uso terapéutico , Seudoxantoma Elástico/tratamiento farmacológico , Seudoxantoma Elástico/genética , Seudoxantoma Elástico/metabolismo , Pirofosfatasas/genética , Pirofosfatasas/metabolismo , Pirofosfatasas/uso terapéutico , Calcificación Vascular/tratamiento farmacológico , Calcificación Vascular/genética

RESUMEN

In a collection of 6 young binturongs (Arctictis binturong), 3 presented with anorexia, lethargy, and footpad swelling diagnosed by surgical biopsy as calcinosis circumscripta. Despite supportive care over the next 4 years, affected binturongs had progressive weight loss, hyperphosphatemia, and hyperkalemia and developed large radiodense deposits in tissues adjacent to appendicular joints, thoracolumbar vertebrae, and the sternum. Two binturongs died and necropsies showed severe periarticular mineral deposition with fibrosis and granulomatous inflammation. Additionally, binturongs had mineralization of distal renal tubules, mineralization of the renal interstitium, and marked mineralization of the pulmonary interstitium and peribronchiolar smooth muscle. Foot biopsies from the surviving binturong were evaluated by spectroscopic microanalysis and were positive for hydroxyapatite, Ca10(PO4)6(OH)2. Periarticular and footpad mineralization in these binturongs is consistent with hydroxyapatite deposition disease, which is rarely described in humans and animals as a primary familial condition or a secondary condition often associated with renal injury.

Asunto(s)

Riñón , Viverridae , Animales , Pie , Hidroxiapatitas , Pulmón

RESUMEN

Extraosseous calcification is a pathologic mineralization process occurring in soft connective tissues (e.g., skin, vessels, tendons, and cartilage). It can take place on a genetic basis or as a consequence of acquired chronic diseases. In this last case, the etiology is multifactorial, including both extra- and intracellular mechanisms, such as the formation of membrane vesicles (e.g., matrix vesicles and apoptotic bodies), mitochondrial alterations, and oxidative stress. This review is an overview of extraosseous calcification mechanisms focusing on the relationships between apoptosis and mineralization in cartilage and vascular tissues, as these are the two tissues mostly affected by a number of age-related diseases having a progressively increased impact in Western Countries.

Asunto(s)

Apoptosis , Calcinosis/patología , Animales , Cartílago/patología , Matriz Extracelular/patología , Humanos , Mitocondrias/patología , Estrés Oxidativo

RESUMEN

PURPOSE: Pseudoxanthoma elasticum (PXE) is a recessive disorder involving skin, eyes and arteries, mainly caused by ABCC6 pathogenic variants. However, almost one fifth of patients remain genetically unsolved despite extensive genetic screening of ABCC6, as illustrated in a large French PXE series of 220 cases. We searched for new PXE gene(s) to solve the ABCC6-negative patients. METHODS: First, family-based exome sequencing was performed, in one ABCC6-negative PXE patient with additional neurological features, and her relatives. CYP2U1, involved in hereditary spastic paraplegia type 56 (SPG56), was selected based on this complex phenotype, and the presence of two candidate variants. Second, CYP2U1 sequencing was performed in a retrospective series of 46 additional ABCC6-negative PXE probands. Third, six additional SPG56 patients were evaluated for PXE skin and eye phenotype. Additionally, plasma pyrophosphate dosage and functional analyses were performed in some of these patients. RESULTS: 6.4% of ABCC6-negative PXE patients (n = 3) harboured biallelic pathogenic variants in CYP2U1. PXE skin lesions with histological confirmation, eye lesions including maculopathy or angioid streaks, and various neurological symptoms were present. CYP2U1 missense variants were confirmed to impair protein function. Plasma pyrophosphate levels were normal. Two SPG56 patients (33%) presented some phenotypic overlap with PXE. CONCLUSION: CYP2U1 pathogenic variants are found in unsolved PXE patients with neurological findings, including spastic paraplegia, expanding the SPG56 phenotype and highlighting its overlap with PXE. The pathophysiology of ABCC6 and CYP2U1 should be explored to explain their respective role and potential interaction in ectopic mineralization.

Asunto(s)

Familia 2 del Citocromo P450/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Seudoxantoma Elástico/genética , Paraplejía Espástica Hereditaria/genética , Calcinosis , Sistema Enzimático del Citocromo P-450/metabolismo , Ojo/patología , Células HEK293 , Humanos , Mutación Missense , Fenotipo , Seudoxantoma Elástico/metabolismo , Seudoxantoma Elástico/patología , Estudios Retrospectivos , Piel/patología , Paraplejía Espástica Hereditaria/metabolismo , Paraplejía Espástica Hereditaria/patología

RESUMEN

Epidemiological studies indicate that elevated alkaline phosphatase activity is associated with increased cardiovascular disease risk. Other epidemiological data demonstrate that mothers giving multiple childbirths (multipara) are also at increased risk of developing late-onset cardiovascular disease. We hypothesized that these two associations stem from a common cause, the insufficient plasma level of the ectopic mineralization inhibitor inorganic pyrophosphate, which is a substrate of alkaline phosphatase. As alkaline phosphatase activity is elevated in pregnancy, we hypothesized that pyrophosphate concentrations decrease gestationally, potentially leading to increased maternal vascular calcification and cardiovascular disease risk in multipara. We investigated plasma pyrophosphate kinetics pre- and postpartum in sheep and at term in humans and demonstrated its shortage in pregnancy, mirroring alkaline phosphatase activity. Next, we tested whether multiparity is associated with increased vascular calcification in pseudoxanthoma elasticum patients, characterized by low intrinsic plasma pyrophosphate levels. We demonstrated that these patients had increased vascular calcification when they give birth multiple times. We propose that transient shortages of pyrophosphate during repeated pregnancies might contribute to vascular calcification and multiparity-associated cardiovascular disease risk threatening hundreds of millions of healthy women worldwide. Future trials are needed to assess if gestational pyrophosphate supplementation might be a suitable prophylactic treatment to mitigate maternal cardiovascular disease risk in multiparous women.

RESUMEN

OBJECTIVE: Pseudoxanthoma elasticum (PXE) is a multisystem heritable disorder caused by mutations in the Abcc6 gene. The disease is characterized by ectopic mineralization of the skin, eyes, and arterial blood vessels. Previous studies have suggested that cardiovascular complications in patients with PXE are caused in part by premature atherosclerosis. The aim of this study is to determine the effect of an atherogenic diet on ectopic mineralization. METHODS: We used Abcc6 tm1JfK mice (Abcc6 -/- mice) as an established preclinical model of PXE. The offspring at age of 4 weeks were divided into two groups and fed the standard control laboratory diet (control group) and the atherogenic diet. Serum lipid profiles and bile acids were measured, and steatosis and tissue mineralization were evaluated by histopathologic analysis and chemical calcium quantification assay, respectively. RESULTS: After 50-58 weeks of feeding an atherogenic diet, the concentrations of total cholesterol, low-density lipoprotein/very-low-density lipoprotein cholesterol, and bile acids were significantly higher in the Abcc6 -/- mice on the atherogenic diet (180.9 ±â€Š14.8 g/L, 145.9 ±â€Š12.9 g/L, and 9.7 ±â€Š1.4 µmol/L, respectively) than in Abcc6 -/- mice on a control diet (85.2 ±â€Š4.8 g/L, 25.1 ±â€Š5.5 g/L, and 3.3 ±â€Š0.5 µmol/L, respectively) (P < 0.001). Hypercholesterolemia was accompanied by extensive lipid accumulation in the liver and aorta, a characteristic feature of steatosis. The direct calcium assay demonstrated significantly increased mineralization of the muzzle skin containing the dermal sheath of vibrissae (57.2 ±â€Š4.4 µmol Ca/gram tissue on the atherogenic diet and 43.9 ±â€Š2.2 µmol Ca/gram tissue on control diet; P < 0.01), a reproducible biomarker of the ectopic mineralization process in these mice. An increased frequency of mineralization was also observed in the kidneys and eyes of mice on the atherogenic diet (P < 0.01). CONCLUSION: These observations suggest that the atherogenic diet caused hypercholesterolemia and accelerated ectopic mineralization in the Abcc6 -/- mice. Our findings have clinical implications for patients with PXE, a currently intractable disorder with considerable morbidity and occasional mortality.

RESUMEN

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.