Histological Findings in the Eyes of Abcc6 Knockout Rat Model of Pseudoxanthoma Elasticum.

Purpose: To describe the ocular findings of murine pseudoxanthoma elasticum (PXE) models with ATP-binding cassette subfamily C member 6 (Abcc6) gene knockout. Methods: This experiment was conducted in four Abcc6-/- rats and compared with six wild-type Abcc6+/+ control rats. The animals underwent necropsy at 6 months of age. Histological examination of the eyes was performed. Results: Histological examination of eight eyes from four Abcc6-/- rats revealed multiple nodular foci of calcification in the uvea, sclera, and conjunctiva, focally in perivascular distribution, as well as linear and nodular calcification of Bruch's membrane. Calcific foci were not associated with inflammation in the knockout rats. There was no evidence of calcification in control eyes. Discussion: The Abcc6-/- rat model shows that PXE can affect multiple ocular tissues beyond the calcification in Bruch's membrane noted in human eyes. Nodular calcific foci probably correspond to comet lesions seen in patients with PXE. The presence of ectopic calcium without inflammation distinguishes it from inflammatory calcium deposition in atherosclerosis. Further studies are needed to determine why PXE does not cause inflammatory infiltration. Translational Relevance: The Abcc6-/- murine model may be suitable for studying ocular PXE pathophysiology and ectopic calcification and developing effective therapies.

Increased Intracranial Arterial Pulsatility and Microvascular Brain Damage in Pseudoxanthoma Elasticum.

BACKGROUND AND PURPOSE: Carotid siphon calcification might contribute to the high prevalence of cerebrovascular disease in pseudoxanthoma elasticum through increased arterial flow pulsatility. This study aimed to compare intracranial artery flow pulsatility, brain volumes, and small-vessel disease markers between patients with pseudoxanthoma elasticum and controls and the association between arterial calcification and pulsatility in pseudoxanthoma elasticum. MATERIALS AND METHODS: Fifty patients with pseudoxanthoma elasticum and 40 age- and sex-matched controls underwent 3T MR imaging, including 2D phase-contrast acquisitions for flow pulsatility in the assessment of ICA and MCA and FLAIR acquisitions for brain volumes, white matter lesions, and infarctions. All patients with pseudoxanthoma elasticum underwent CT scanning to measure siphon calcification. Flow pulsatility (2D phase-contrast), brain volumes, white matter lesions, and infarctions (3D T1 and 3D T2 FLAIR) were compared between patients and controls. The association between siphon calcification and pulsatility in pseudoxanthoma elasticum was tested with linear regression models. RESULTS: Patients with pseudoxanthoma elasticum (mean age, 57 [SD, 12] years; 24 men) had significantly higher pulsatility indexes (1.05; range, 0.94-1.21 versus 0.94; range, 0.82-1.04; P = .02), lower mean GM volumes (597 [SD, 53] mL versus 632 [SD, 53] mL; P < .01), more white matter lesions (2.6; range, 0.5-7.5 versus 1.1; range, 0.5-2.4) mL; P = .05), and more lacunar infarctions (64 versus 8, P = .04) than controls (mean age, 58 [SD, 11] years; 20 men). Carotid siphon calcification was associated with higher pulsatility indexes in patients with pseudoxanthoma elasticum (ß = 0.10; 95% CI, 0.01-0.18). CONCLUSIONS: Patients with pseudoxanthoma elasticum have increased intracranial artery flow pulsatility and measures of small-vessel disease. Carotid siphon calcification might underlie the high prevalence of cerebrovascular disease in pseudoxanthoma elasticum.

Pseudoxanthoma-elasticum-like changes on the soft palate.

Pseudoxanthoma elasticum (PXE) is an autosomal recessive genetic disorder characterized by aberrant fragmentation and calcification of elastic fibers, leading to characteristic cutaneous, ophthalmic, and cardiovascular manifestations. PXE demonstrates significant phenotypic variability; involvement of the oral mucosa may be the only clue to the diagnosis. Reports on mucous membrane involvement in PXE are scarce. Here, we present a case of PXE-like changes in the oral cavity. A 70-year-old male patient presented with a painless leukoplakic lesion on the soft palate. Biopsy revealed numerous degenerated fibers in the lamina propria. Verhoeff-van Gieson and von Kossa staining confirmed their identity as calcified elastic fibers. A histopathological diagnosis of PXE-like changes was made; the patient was referred to ophthalmology where angioid streaks were visualized fundoscopically. PXE-like changes in the absence of the characteristic genetic mutation have also been reported with or without systemic manifestations. Furthermore, PXE-like changes have been reported in up to 10% of oral biopsy specimens undertaken without clinical suspicion for PXE. Therefore, the significance of such changes in isolation is unclear. Clinicians and pathologists should be aware of the potential oral manifestations of PXE to facilitate prompt diagnosis and subspecialist referral.

The Consideration of Pseudoxanthoma Elasticum as a Progeria Syndrome.

BACKGROUND: Pseudoxanthoma elasticum (PXE) is a rare autosomal recessive disorder caused by mutations in the ATP-binding cassette sub-family C member 6 (ABCC6) gene. Patients with PXE show molecular and clinical characteristics of known premature aging syndromes, such as Hutchinson-Gilford progeria syndrome (HGPS). Nevertheless, PXE has only barely been discussed against the background of premature aging, although a detailed characterization of aging processes in PXE could contribute to a better understanding of its pathogenesis. Thus, this study was performed to evaluate whether relevant factors which are known to play a role in accelerated aging processes in HGPS pathogenesis are also dysregulated in PXE. METHODS: Primary human dermal fibroblasts from healthy donors (n = 3) and PXE patients (n = 3) and were cultivated under different culture conditions as our previous studies point towards effects of nutrient depletion on PXE phenotype. Gene expression of lamin A, lamin C, nucleolin, farnesyltransferase and zinc metallopeptidase STE24 were determined by quantitative real-time polymerase chain reaction. Additionally, protein levels of lamin A, C and nucleolin were evaluated by immunofluorescence and the telomere length was analyzed. RESULTS: We could show a significant decrease of lamin A and C gene expression in PXE fibroblasts under nutrient depletion compared to controls. The gene expression of progerin and farnesyltransferase showed a significant increase in PXE fibroblasts when cultivated in 10% fetal calf serum (FCS) compared to controls. Immunofluorescence microscopy of lamin A/C and nucleolin and mRNA expression of zinc metallopeptidase STE24 and nucleolin showed no significant changes in any case. The determination of the relative telomere length showed significantly longer telomeres for PXE fibroblasts compared to controls when cultivated in 10% FCS. CONCLUSIONS: These data indicate that PXE fibroblasts possibly undergo a kind of senescence which is independent of telomere damage and not triggered by defects of the nuclear envelope or nucleoli deformation.

Correlation of systemic involvement and presence of pathological skin calcification assessed by ex vivo nonlinear microscopy in Pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE (OMIM 264800)) is an autosomal recessive connective tissue disorder mainly caused by mutations in the ABCC6 gene. PXE results in ectopic calcification primarily in the skin, eye and blood vessels that can lead to blindness, peripheral arterial disease and stroke. Previous studies found correlation between macroscopic skin involvement and severe ophthalmological and cardiovascular complications. This study aimed to investigate correlation between skin calcification and systemic involvement in PXE. Ex vivo nonlinear microscopy (NLM) imaging was performed on formalin fixed, deparaffinized, unstained skin sections to assess the extent of skin calcification. The area affected by calcification (CA) in the dermis and density of calcification (CD) was calculated. From CA and CD, calcification score (CS) was determined. The number of affected typical and nontypical skin sites were counted. Phenodex + scores were determined. The relationship between the ophthalmological, cerebro- and cardiovascular and other systemic complications and CA, CD and CS, respectively, and skin involvement were analyzed. Regression models were built for adjustment to age and sex. We found significant correlation of CA with the number of affected typical skin sites (r = 0.48), the Phenodex + score (r = 0.435), extent of vessel involvement (V-score) (r = 0.434) and disease duration (r = 0.48). CD correlated significantly with V-score (r = 0.539). CA was significantly higher in patients with more severe eye (p = 0.04) and vascular (p = 0.005) complications. We found significantly higher CD in patients with higher V-score (p = 0.018), and with internal carotid artery hypoplasia (p = 0.045). Significant correlation was found between higher CA and the presence of macula atrophy (ß = - 0.44, p = 0.032) and acneiform skin changes (ß = 0.40, p = 0.047). Based on our results, the assessment of skin calcification pattern with nonlinear microscopy in PXE may be useful for clinicians to identify PXE patients who develop severe systemic complications.

The pathogenic c.1171A>G (p.Arg391Gly) and c.2359G>A (p.Val787Ile) ABCC6 variants display incomplete penetrance causing pseudoxanthoma elasticum in a subset of individuals.

ABCC6 promotes ATP efflux from hepatocytes to bloodstream. ATP is metabolized to pyrophosphate, an inhibitor of ectopic calcification. Pathogenic variants of ABCC6 cause pseudoxanthoma elasticum, a highly variable recessive ectopic calcification disorder. Incomplete penetrance may initiate disease heterogeneity, hence symptoms may not, or differently manifest in carriers. Here, we investigated whether incomplete penetrance is a source of heterogeneity in pseudoxanthoma elasticum. By integrating clinical and genetic data of 589 patients, we created the largest European cohort. Based on allele frequency alterations, we identified two incomplete penetrant pathogenic variants, c.2359G>A (p.Val787Ile) and c.1171A>G (p.Arg391Gly), with 6.5% and 2% penetrance, respectively. However, when penetrant, the c.1171A>G (p.Arg391Gly) manifested a clinically unaltered severity. After applying in silico and in vitro characterization, we suggest that incomplete penetrant variants are only deleterious if a yet unknown interacting partner of ABCC6 is mutated simultaneously. The low penetrance of these variants should be contemplated in genetic counseling.

Seeing through the cracks.

Pseudoxanthoma elasticum (PXE) is an autosomal recessive multisystem disorder showing phenotypic heterogeneity giving rise to complex comorbidities. The most 'visible' signs are dermatological; however, these may be subtle and hidden from the view of an affected individual. Ophthalmic signs can be easily missed, and here we highlight the importance of a multisystem assessment. We report a patient who developed advanced sight loss due to maculopathy whose underlying PXE aetiology went unnoticed until subtle skin signs were noticed on the lateral aspect of his neck. He was aware of the skin changes. Careful review of his previous retinal imaging showed the presence of 'angioid streaks' and anatomic alteration at the outer retina-Bruch membrane associated with his prior history of choroidal neovascularisation. The diagnosis was subsequently confirmed by skin biopsy and genetic testing. This case highlights the subtlety of both dermatological and ophthalmic signs in PXE.

Cutaneous vascular calcification. Perieccrine calcification as a diagnostic key for calciphylaxis.

BACKGROUND: Attempts have been made to establish discriminative criteria between classic calciphylaxis (CPX) and those cases in which cutaneous vascular calcification (CVC) represents an incidental finding (epiphenomenon). METHODS: Retrospective, observational cohort study of patients with CVC to distinguish clinicopathological features between CVC as classic CPX (CVC in cutaneous lesions with erythematous-violaceous plaques with or without ulceration) or as an epiphenomenon (CVC in cutaneous lesions with known diagnosis). Different clinicopathological parameters and the presence of perieccrine calcification and pseudoxanthoma elasticum (PXE)-like changes were evaluated. RESULTS: Sixty-six patients were studied. The CPX group showed a significantly higher percentage of renal failure, hypertension, altered laboratory parameters, painful lesions, and mortality rate. Histopathologically, the CPX group was associated with more than one vessel per field involved with subintimal concentric calcification and perieccrine calcification (observed exclusively in the CPX group), while PXE-like changes, although more frequent in the CPX group, were also observed in the epiphenomenon group. CONCLUSION: Perieccrine calcification and the presence of more than one vessel per field involved by concentric pattern calcification could be used as a diagnostic marker of CPX. Although PXE-like changes are not an exclusive marker, they could suggest CPX diagnosis.

Inherited connective tissue diseases highlight macromolecular network interdependences in skin extracellular matrix: a histomorphometric study.

Mutation of just a single extracellular matrix protein, a receptor or enzyme involved in connective tissue metabolism is sufficient to cause systemic pathologies and failure of tissues that are subjected to strong mechanical stresses. Skin histological and computerized image analyses can provide a good qualitative and quantitative indication of these inherited connective tissue diseases. In this study, skin biopsies from young (10 to 25 years) and middle-aged patients (26 to 50 years) suffering from Ehlers-Danlos syndromes (EDS), Marfan syndrome (MS) or pseudoxanthoma elasticum (PXE) were studied after specific staining of both the collagen and elastic networks. Findings from the histomorphometric analyses conducted on skin sections of the patients with EDS, MS and PXE were compared to skin sections of healthy subjects from the same age groups. Our results show that both the collagen and the elastic networks were affected in all the studied pathological cases, but that the adverse changes to the elastic network in older patients were distinct from the physiological changes observed during aging process for healthy subjects. This degenerative process may be explained by an added phenomenon involving a general connective tissue proteolysis.

Inhibition of the DNA Damage Response Attenuates Ectopic Calcification in Pseudoxanthoma Elasticum.

Pseudoxanthoma elasticum (PXE) is a heritable ectopic calcification disorder with multiorgan clinical manifestations. The gene at default, ABCC6, encodes an efflux transporter, ABCC6, which is a critical player regulating the homeostasis of inorganic pyrophosphate, a potent endogenous anticalcification factor. Previous studies suggested that systemic inorganic pyrophosphate deficiency is the major but not the exclusive cause of ectopic calcification in PXE. In this study, we show that the DNA damage response (DDR) and poly(ADP-ribose) (PAR) pathways are involved locally in PXE at sites of ectopic calcification. Genetic inhibition of PAR polymerase 1 gene PARP1, the predominant PAR-producing enzyme, showed a 54% reduction of calcification in the muzzle skin in Abcc6‒/‒Parp1‒/‒ mice, compared with that of age-matched Abcc6‒/‒Parp1+/+ littermates. Subsequently, oral administration of minocycline, an inhibitor of DDR/PAR signaling, resulted in an 86% reduction of calcification in the muzzle skin of Abcc6‒/‒ mice. Minocycline treatment also attenuated the DDR/PAR signaling and reduced the calcification of dermal fibroblasts derived from patients with PXE. The anticalcification effect of DDR/PAR inhibition was not accompanied by alterations in plasma inorganic pyrophosphate concentrations. These results suggest that local DDR/PAR signaling in calcification-prone tissues contributes to PXE pathogenesis and that its inhibition might provide a promising treatment strategy for ectopic calcification in PXE, a currently intractable disease.

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.

Comprehensive validation of a diagnostic strategy for sequencing genes with one or multiple pseudogenes using pseudoxanthoma elasticum as a model.

Pseudogenes are frequently encountered noncoding sequences with a high sequence similarity to their protein-coding paralogue. For this reason, their presence is often considered troublesome in molecular diagnostics. In pseudoxanthoma elasticum (PXE), a disease predominantly caused by mutations in ATP-binding cassette family C member 6 (ABCC6), the presence of two pseudogenes complicates the analysis of sequence data. With whole-exome sequencing (WES) becoming the standard of care in molecular diagnostics, we wanted to evaluate whether this technique is as reliable as gene-specific targeted enrichment analysis for the analysis of ABCC6. We established a PCR-based targeted enrichment and next-generation sequencing testing approach and demonstrated that the ABCC6-specific enrichment combined with the applied mapping algorithm overcomes the complication of ABCC6 pseudogene aspecificities, contrary to WES. We propose a time- and cost-efficient diagnostic strategy for comprehensive and accurate molecular genetic testing of PXE, which is highly automatable.

Fast visual exploration of mass spectrometry images with interactive dynamic spectral similarity pseudocoloring.

Mass Spectrometry Imaging (MSI) is an established and still evolving technique for the spatial analysis of molecular co-location in biological samples. Nowadays, MSI is expanding into new domains such as clinical pathology. In order to increase the value of MSI data, software for visual analysis is required that is intuitive and technique independent. Here, we present QUIMBI (QUIck exploration tool for Multivariate BioImages) a new tool for the visual analysis of MSI data. QUIMBI is an interactive visual exploration tool that provides the user with a convenient and straightforward visual exploration of morphological and spectral features of MSI data. To improve the overall quality of MSI data by reducing non-tissue specific signals and to ensure optimal compatibility with QUIMBI, the tool is combined with the new pre-processing tool ProViM (Processing for Visualization and multivariate analysis of MSI Data), presented in this work. The features of the proposed visual analysis approach for MSI data analysis are demonstrated with two use cases. The results show that the use of ProViM and QUIMBI not only provides a new fast and intuitive visual analysis, but also allows the detection of new co-location patterns in MSI data that are difficult to find with other methods.

Pseudoxanthoma elasticum overlaps hereditary spastic paraplegia type 56.

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.

Linking ABCC6 Deficiency in Primary Human Dermal Fibroblasts of PXE Patients to p21-Mediated Premature Cellular Senescence and the Development of a Proinflammatory Secretory Phenotype.

Pseudoxanthoma elasticum (PXE) is a rare autosomal-recessive disorder that is mainly caused by mutations in the ATP-binding cassette sub-family C member 6 (ABCC6) gene. Clinically PXE is characterized by a loss of skin elasticity, arteriosclerosis or visual impairments. It also shares some molecular characteristics with known premature aging syndromes like the Hutchinson-Gilford progeria syndrome (HGPS). However, little is known about accelerated aging processes, especially on a cellular level for PXE now. Therefore, this study was performed to reveal a potential connection between premature cellular aging and PXE pathogenesis by analyzing cellular senescence, a corresponding secretory phenotype and relevant factors of the cell cycle control in primary human dermal fibroblasts of PXE patients. Here, we could show an increased senescence-associated ß-galactosidase (SA-ß-Gal) activity as well as an increased expression of proinflammatory factors of a senescence-associated secretory phenotype (SASP) like interleukin 6 (IL6) and monocyte chemoattractant protein-1 (MCP1). We further observed an increased gene expression of the cyclin-dependent kinase inhibitor (CDKI) p21, but no simultaneous induction of p53 gene expression. These data indicate that PXE is associated with premature cellular senescence, which is possibly triggered by a p53-independent p21-mediated mechanism leading to a proinflammatory secretory phenotype.