Pseudoxanthoma Elasticum Phenotype Research Articles

The Consideration of Pseudoxanthoma Elasticum as a Progeria Syndrome.

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. 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. 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. 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.

Inorganic Pyrophosphate Plasma Levels Are Decreased in Pseudoxanthoma Elasticum Patients and Heterozygous Carriers but Do Not Correlate with the Genotype or Phenotype.

Pseudoxanthoma elasticum (PXE) is a rare ectopic calcification disorder affecting soft connective tissues that is caused by biallelic ABCC6 mutations. While the underlying pathomechanisms are incompletely understood, reduced circulatory levels of inorganic pyrophosphate (PPi)-a potent mineralization inhibitor-have been reported in PXE patients and were suggested to be useful as a disease biomarker. In this study, we explored the relation between PPi, the ABCC6 genotype and the PXE phenotype. For this, we optimized and validated a PPi measurement protocol with internal calibration that can be used in a clinical setting. An analysis of 78 PXE patients, 69 heterozygous carriers and 14 control samples revealed significant differences in the measured PPi levels between all three cohorts, although there was overlap between all groups. PXE patients had a ±50% reduction in PPi levels compared to controls. Similarly, we found a ±28% reduction in carriers. PPi levels were found to correlate with age in PXE patients and carriers, independent of the ABCC6 genotype. No correlations were found between PPi levels and the Phenodex scores. Our results suggest that other factors besides PPi are at play in ectopic mineralization, which limits the use of PPi as a predictive biomarker for severity and disease progression.

Rare Modifier Variants Alter the Severity of Cardiovascular Disease in Pseudoxanthoma Elasticum: Identification of Novel Candidate Modifier Genes and Disease Pathways Through Mixture of Effects Analysis.

Introduction: Pseudoxanthoma elasticum (PXE), an ectopic mineralization disorder caused by pathogenic ABCC6 variants, is characterized by skin, ocular and cardiovascular (CV) symptoms. Due to striking phenotypic variability without genotype-phenotype correlations, modifier genes are thought to play a role in disease variability. In this study, we evaluated the collective modifying effect of rare variants on the cardiovascular phenotype of PXE.Materials and Methods: Mixed effects of rare variants were assessed by Whole Exome Sequencing in 11 PXE patients with an extreme CV phenotype (mild/severe). Statistical analysis (SKAT-O and C-alpha testing) was performed to identify new modifier genes for the CV PXE phenotype and enrichment analysis for genes significantly associated with the severe cohort was used to evaluate pathway and gene ontology features.Results Respectively 16 (SKAT-O) and 74 (C-alpha) genes were significantly associated to the severe cohort. Top significant genes could be stratified in 3 groups–calcium homeostasis, association with vascular disease and induction of apoptosis. Comparative analysis of both analyses led to prioritization of four genes (NLRP1, SELE, TRPV1, and CSF1R), all signaling through IL-1B.Conclusion This study explored for the first time the cumulative effect of rare variants on the severity of cardiovascular disease in PXE, leading to a panel of novel candidate modifier genes and disease pathways. Though further validation is essential, this panel may aid in risk stratification and genetic counseling of PXE patients and will help to gain new insights in the PXE pathophysiology.

Statins as a Therapeutic Approach for the Treatment of Pseudoxanthoma Elasticum Patients: Evaluation of the Spectrum Efficacy of Atorvastatin In Vitro

Pseudoxanthoma elasticum (PXE) is an autosomal recessive disorder caused by mutations in the ATP-binding cassette sub-family C member 6 gene. Our previous studies revealed that PXE might be associated with premature aging. Treatment with statins showed positive effects not only for PXE but also for other diseases associated with premature aging like Hutchinson–Gilford progeria syndrome. Nevertheless, the molecular mechanisms in the case of PXE remain unclear. Thus, this study was performed to evaluate the efficiency of atorvastatin by analyzing key characteristics of the PXE phenotype in primary human dermal fibroblasts of PXE patients. Our data indicate that an atorvastatin treatment has a positive effect, especially on factors associated with cholesterol biosynthesis and prenylation processes, whereas the effect on age- and calcification-related factors was less pronounced.

Whole exome sequencing identified three ABCC6 variants in two Pakistani families with pseudoxanthoma elasticum phenotype.

Pseudoxanthoma elasticum (PXE) is an autosomal recessive disorder of ectopic mineralization and fragmentation of elastic fibers in skin, eyes, cardiovascular and digestive system. PXE is caused by sequence variants in ABCC6, which encodes multidrug resistance-associated protein 6 (MRP6, also known as the ABCC6 protein). MRP6 is an important regulator of inorganic plasma pyrophosphate that acts as an inhibitor of ectopic mineralization observed in PXE patients with low inorganic plasma pyrophosphate levels. The current study was designed to investigate underlying genetic defect in two unrelated Pakistani families affected with PXE. Whole exome sequencing followed by Sanger sequencing was performed to identify causative variants. A novel homozygous frameshift variant (c.1799_1805dupGTCTGGT) was identified in one family and two previously reported missense variants (c.2294G > A and c.2974G > A) in compound heterozygous form in the other family. We identified ABCC6 variants that are likely cause of the PXE disease in the tested families. Genetic analysis of these families could be useful for pre-symptomatic diagnosis and genetic counselling of the affected families.

Acquired pseudoxanthoma elasticum phenotype in a patient with hemoglobin Southampton, a rare and severe hemoglobinopathy

Acquired pseudoxanthoma elasticum phenotype in a patient with hemoglobin Southampton, a rare and severe hemoglobinopathy

Copy number variation in the ATP-binding cassette transporter ABCC6 gene and ABCC6 pseudogenes in patients with pseudoxanthoma elasticum.

Single mutations in the ATP-binding cassette transporter (ABCC6) gene (OMIM 603234) are known to cause the rare autosomal recessive disease pseudoxanthoma elasticum (PXE). Recently, we have found that copy number variations (CNVs) in pseudogenes of the ABCC6 gene are quite common. The aim of this study was to investigate the frequency and possible contribution of CNV in ABCC6 and its pseudogenes in PXE. Genomic DNA from 212 PXE individuals were examined for copy number by pyrosequencing and quantitative polymerase chain reaction (PCR) and compared with healthy individuals. The frequency of PXE individuals with any CNV was higher than in healthy individuals. The majority of variation comprised known and possibly new deletions in the ABCC6 gene and duplications of the ABCC6P1 and ABCC6P2 genes. ABCC6 deletions and ABCC6P2 duplications were not observed in 142 healthy individuals. In conclusion, by pyrosequencing and quantitative PCR, we were able to detect known and possibly new deletions in the ABCC6 gene that may have caused the PXE phenotype. Pyrosequencing may be used in PXE patients who have obtained incomplete genotype from conventional techniques. The frequency of ABCC6P2 pseudogene duplication was more common in PXE patients than healthy individuals and may affect the PXE phenotype.

The vascular phenotype in Pseudoxanthoma elasticum and related disorders: contribution of a genetic disease to the understanding of vascular calcification

Vascular calcification is a complex and dynamic process occurring in various physiological conditions such as aging and exercise or in acquired metabolic disorders like diabetes or chronic renal insufficiency. Arterial calcifications are also observed in several genetic diseases revealing the important role of unbalanced or defective anti- or pro-calcifying factors. Pseudoxanthoma elasticum (PXE) is an inherited disease (OMIM 264800) characterized by elastic fiber fragmentation and calcification in various soft conjunctive tissues including the skin, eyes, and arterial media. The PXE disease results from mutations in the ABCC6 gene, encoding an ATP-binding cassette transporter primarily expressed in the liver, kidneys suggesting that it is a prototypic metabolic soft-tissue calcifying disease of genetic origin. The clinical expression of the PXE arterial disease is characterized by an increased risk for coronary (myocardial infarction), cerebral (aneurysm and stroke), and lower limb peripheral artery disease. However, the structural and functional changes in the arterial wall induced by PXE are still unexplained. The use of a recombinant mouse model inactivated for the Abcc6 gene is an important tool for the understanding of the PXE pathophysiology although the vascular impact in this model remains limited to date. Overlapping of the PXE phenotype with other inherited calcifying diseases could bring important informations to our comprehension of the PXE disease.

Coordinated orphan disease research: yes, we can!

Coordinated orphan disease research: yes, we can!

Administration of Bone Marrow Derived Mesenchymal Stem Cells into the Liver: Potential to Rescue Pseudoxanthoma Elasticum in a Mouse Model (Abcc6−/−)

Pseudoxanthoma elasticum (PXE) is a heritable ectopic mineralization disorder caused by loss-of-function mutations in the ABCC6 gene which is primarily expressed in the liver. There is currently no effective treatment for PXE. In this study, we characterized bone marrow derived mesenchymal stem cells (MSCs) and evaluated their ability to contribute to liver regeneration, with the aim to rescue PXE phenotype. The MSCs, isolated from GFP-transgenic mice by magnetic cell sorting, were shown to have high potential for hepatic differentiation, with expression of Abcc6, in culture. These cells were transplanted into the livers of 4-week-old immunodeficient Abcc6−/− mice by intrasplenic injection one day after partial hepatectomy, when peak expression of the stromal cell derived factor-1 (SDF-1) in the liver was observed. Fluorescent bioimaging analyses indicated that transplanted MSCs homed into liver between day 1 and 7, and significant numbers of GFP-positive cells were confirmed in the liver by immunofluorescence. Moreover, enhanced engraftment efficiency was observed with MSCs with high expression levels of the chemokine receptor Cxcr4, a receptor for SDF-1. These data suggest that purified MSCs have the capability of differentiating into hepatic lineages relevant to PXE pathogenesis and may contribute to partial correction of the PXE phenotype.

Histological skin changes in heterozygote carriers of mutations in ABCC6, the gene causing pseudoxanthoma elasticum

Pseudoxanthoma elasticum (PXE) is related to mutations in the ABCC6 gene and characterized pathologically by dystrophic and mineralized elastic fibres. Heterozygote carriers of ABCC6 mutations may have a limited PXE phenotype. To compare histological changes in the skin of genotyped siblings from two PXE pedigrees. Mutation analysis of ABCC6 was performed. Skin biopsy samples were stained (orcein) and immunolabelled for elastin, and for vitronectin and bone sialoprotein, which are partially responsible for the mineralization within the elastorrhexic fibres. Results In all individuals mutation analysis of ABCC6 allowed definition of the genotype status, i.e. PXE (n = 2), heterozygote (n = 7) or wild type (n = 2). The study identified three histological phenotypes related to the ABCC6 genotype in siblings from both families. Heterozygote carriers had changes in dermal elastic fibre organization, morphology and labelling midway between those seen in PXE skin and normal skin. Even though the number of individuals studied here is small and precludes any hasty generalization, having a single mutation in the ABCC6 gene seems enough to modify dermal elastic fibres. The relevance of performing a skin biopsy to identify heterozygote carriers in the family of a PXE patient remains to be determined.

Augmentation de l’hémoglobine A 2 au cours du pseudoxanthome élastique

Pseudoxanthoma elasticum (PXE) is normally associated with mutations in the ABCC6 gene. A PXE phenotype without mutations in ABCC6 has been described in Greek and Italian patients presenting with beta thalassemia. We attempted to determine the incidence of beta thalassemia in a cohort of French patients with PXE.Fifty patients with PXE were included in the study. Laboratory examinations comprised hemoglobin electrophoresis, ABCC6 gene study and in some studies: mutation analysis, beta-globin gene.No cases of beta thalassemia were diagnosed in this cohort of French patients with PXE. However, 20% of the latter exhibited a significant but isolated (i.e. without microcytic anemia) increase of hemoglobin A2 (HbA2). Statistical comparisons showed no difference in terms of geographical origin or severity of PXE between patients with high levels of HbA2 and those with normal levels of HbA2 other than the extent of cutaneous involvement. Study of the beta-globin gene displayed mutations only in the two patients with the highest recorded levels of HbA2. ABCC6 + beta-globin digenism was ruled out of the pathogenesis of PXE.The PXE phenotype seen in some patients with beta thalassemia appears to be associated with epigenetic modification of ABCC6 transcription and depends specifically on the beta globin locus. Isolated increase in HbA2 is probably a laboratory marker for PXE. Here again, a functional epigenetic reaction between ABCC6 and the beta-globin locus was suspected. However, these reciprocal interactions are clearly unequal since the change in ABCC6 transcription occurring during the course of beta thalassaemia is responsible for a PXE phenotype while increased HbA2 during the course of PXE has no clinical consequences.

HNF4α and NF-E2 are key transcriptional regulators of the murine Abcc6 gene expression

Mutations in an ABC transporter gene called ABCC6 are responsible for pseudoxanthoma elasticum (PXE), a rare heritable disease characterized by elastic fiber calcification in skin, ocular and vascular tissues. The presumed function of this ABC transporter is to export metabolites from polarized cells. However, the endogenous substrate(s) are unknown and the exact relationship with elastic fibers is unclear. As ABCC6 is only expressed at high level in liver and kidneys, tissues seemingly unrelated to the PXE phenotype, we explored the transcriptional regulation of the murine Abcc6 gene to define the transcriptional signal conferring tissue specificity and to gather clues on its possible biological function. We cloned 2.9 kb of the mAbcc6 5'-flanking region and several deletion constructs linked to a luciferase reporter gene. We delineated a proximal promoter and a liver-specific enhancer region. We also demonstrated that the proximal region is a TATA-less promoter requiring an intact CCAAT-box and Sp1 binding for its basal activity. By using reporter assays and chromatin immunoprecipitations, we showed that HNF4alpha and surprisingly, NF-E2, enhanced the mAbcc6 promoter activity. The involvement of both HNF4alpha and NF-E2 in the mAbcc6 gene regulation suggests that Abcc6 might be involved in a detoxification processes related to hemoglobin or heme.

Pseudoxanthoma Elasticum: the End of the Autosomal Dominant Segregation Myth

Pseudoxanthoma elasticum (PXE) is a heritable connective-tissue disorder affecting the eye, skin, and vascular system. Recent publications show that PXE exclusively segregates in an autosomal recessive fashion. However, the lack of an internationally accepted clinical "gold standard" for PXE, our incomplete knowledge of PXE etiology, and the incomplete nature of some molecular, clinical, and environmental studies warrant further investigation.

Molecular genetics of pseudoxanthoma elasticum: type and frequency of mutations inABCC6

Pseudoxanthoma elasticum (PXE) is a systemic heritable disorder that affects the elastic tissue in the skin, eye, and cardiovascular system. Mutations in the ABCC6 gene cause PXE. We performed a mutation screen in ABCC6 using haplotype analysis in conjunction with direct sequencing to achieve a mutation detection rate of 97%. This screen consisted of 170 PXE chromosomes in 81 families, and detected 59 distinct mutations (32 missense, eight nonsense, and six likely splice-site point mutations; one small insertion; and seven small and five large deletions). Forty-three of these mutations are novel variants, which increases the total number of PXE mutations to 121. While most mutations are rare, three nonsense mutations, a splice donor site mutation, and the large deletion comprising exons 23-29 (c.2996_4208del) were identified as relatively frequent PXE mutations at 26%, 5%, 3.5%, 3%, and 11%, respectively. Chromosomal haplotyping with two proximal and two distal polymorphic markers flanking ABCC6 demonstrated that most chromosomes that carry these relatively frequent PXE mutations have related haplotypes specific for these mutations, which suggests that these chromosomes originate from single founder mutations. The types of mutations found support loss-of-function as the molecular mechanism for the PXE phenotype. In 76 of the 81 families, the affected individuals were either homozygous for the same mutation or compound heterozygous for two mutations. In the remaining five families with one uncovered mutation, affected showed allelic compound heterozygosity for the cosegregating PXE haplotype. This demonstrates pseudo-dominance as the relevant inheritance mechanism, since disease transmission to the next generation always requires one mutant allelic variant from each parent. In contrast to other previous clinical and molecular claims, our results show evidence only for recessive PXE. This has profound consequences for the genetic counseling of families with PXE.

Patients with premature coronary artery disease who carry the ABCC6 R1141X mutation have no Pseudoxanthoma Elasticum phenotype

Background Pseudoxanthoma elasticum (PXE) is an inherited disorder of elastic tissue. We recently found that heterozygosity for the frequent (0.8% prevalence in Dutch population) R1141X mutation in the PXE gene coding for the ABCC6 transporter, is associated with a fourfold risk of premature coronary artery disease. Yet, it is not clear whether or not heterozygosity for this mutation results in a mild PXE phenotype. The objective of our study was to determine if skin and/or eye abnormalities related to a PXE phenotype could be found in patients with premature coronary artery disease, with and without the R1141X mutation. Methods R1141X mutation carriers with premature coronary artery disease (cases) and patients with premature coronary artery disease with no–or not known–mutation (controls) were studied. Cases and controls were examined for PXE-like skin changes and retinal angioid streaks, peau d'orange or pigment epithelium changes. Results 7 cases and 31 controls were analysed. In both the mutation-positive and the control group, skin inspection and eye fundus examination did not reveal any dermatological or ocular signs of PXE. Conclusions Carriers for the ABCC6 R1141X mutation, which is frequent and confers a high risk of premature coronary artery disease, do not commonly have skin or eye abnormalities consistent with a mild PXE phenotype.

Analysis of the frequent R1141X mutation in the ABCC6 gene in pseudoxanthoma elasticum.

To characterize the ABCC6 R1141X nonsense mutation, which is implicated in more than 25% of a cohort of patients from The Netherlands with pseudoxanthoma elasticum (PXE). A combination of single-strand conformational polymorphism (SSCP), PCR, sequencing, and Southern blot analysis was used to identify mutations in the ABCC6 gene in 62 patients. Haplotypes of 16 patients with the R1141X mutation were determined with eight polymorphic markers spanning the ABCC6 locus. The effect of the R1141X mutation on the expression of ABCC6 was studied in leukocytes and cultured dermal fibroblasts from affected skin in patients heterozygous or homozygous for the R1141X mutation. ABCC6 expression was analyzed by RT-PCR and immunocytochemistry with ABCC6-specific monoclonal antibodies. The ABCC6 R1141X mutation was found on 19 alleles in 16 patients with PXE and occurred in heterozygous, homozygous, or compound heterozygous form. All R1141X alleles were associated with a common haplotype, covering at least three intragenic ABCC6 markers. None of the patients or healthy control subjects had a similar ABCC6 haplotype. Furthermore, the results showed that the expression of the normal allele in R1141X heterozygotes was predominant, whereas no detectable, or very low, ABCC6 mRNA levels were found in R1141X homozygotes. Immunocytochemical staining of cultured dermal fibroblasts with ABCC6-specific monoclonal antibodies showed no evidence of the presence of a truncated protein in patients with PXE who were homozygous for R1141X. A specific founder effect for the R1141X mutation exists in Dutch patients with PXE. The R1141X mutation induces instability of the aberrant mRNA. Functional haploinsufficiency or loss of function of ABCC6 caused by mechanisms, such as nonsense-mediated decay (NMD), may be involved in the PXE phenotype.

Loss of ATP-dependent Transport Activity in Pseudoxanthoma Elasticum-associated Mutants of Human ABCC6 (MRP6)

Mutations in the ABCC6 (MRP6) gene cause pseudoxanthoma elasticum (PXE), a rare heritable disorder resulting in the calcification of elastic fibers. In the present study a cDNA encoding a full-length normal variant of ABCC6 was amplified from a human kidney cDNA library, and the protein was expressed in Sf9 insect cells. In isolated membranes ATP binding as well as ATP-dependent active transport by ABCC6 was demonstrated. We found that glutathione conjugates, including leukotriene C(4) and N-ethylmaleimide S-glutathione (NEM-GS), were actively transported by human ABCC6. Organic anions (probenecid, benzbromarone, indomethacin), known to interfere with glutathione conjugate transport of human ABCC1 and ABCC2, inhibited the ABCC6-mediated NEM-GS transport in a specific manner, indicating that ABCC6 has a unique substrate specificity. We have also expressed three missense mutant forms of ABCC6, which have recently been shown to cause PXE. MgATP binding was normal in these proteins; ATP-dependent NEM-GS or leukotriene C(4) transport, however, was abolished. Our data indicate that human ABCC6 is a primary active transporter for organic anions. In the three ABCC6 mutant forms examined, the loss of transport activity suggests that these mutations result in a PXE phenotype through a direct influence on the transport activity of this ABC transporter.

A Spectrum of ABCC6 Mutations Is Responsible for Pseudoxanthoma Elasticum

To better understand the pathogenetics of pseudoxanthoma elasticum (PXE), we performed a mutational analysis of ATP-binding cassette subfamily C member 6 (ABCC6) in 122 unrelated patients with PXE, the largest cohort of patients yet studied. Thirty-six mutations were characterized, and, among these, 28 were novel variants (for a total of 43 PXE mutations known to date). Twenty-one alleles were missense variants, six were small insertions or deletions, five were nonsense, two were alleles likely to result in aberrant mRNA splicing, and two were large deletions involving ABCC6. Although most mutations appeared to be unique variants, two disease-causing alleles occurred frequently in apparently unrelated individuals. R1141X was found in our patient cohort at a frequency of 18.8% and was preponderant in European patients. ABCC6del23-29 occurred at a frequency of 12.9% and was prevalent in patients from the United States. These results suggested that R1141X and ABCC6del23-29 might have been derived regionally from founder alleles. Putative disease-causing mutations were identified in approximately 64% of the 244 chromosomes studied, and 85.2% of the 122 patients were found to have at least one disease-causing allele. Our results suggest that a fraction of the undetected mutant alleles could be either genomic rearrangements or mutations occurring in noncoding regions of the ABCC6 gene. The distribution pattern of ABCC6 mutations revealed a cluster of disease-causing variants within exons encoding a large C-terminal cytoplasmic loop and in the C-terminal nucleotide-binding domain (NBD2). We discuss the potential structural and functional significance of this mutation pattern within the context of the complex relationship between the PXE phenotype and the function of ABCC6.

Mutations in a gene encoding an ABC transporter cause pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE) is a heritable disorder characterized by calcification of elastic fibres in skin, arteries and retina that results in dermal lesions with associated laxity and loss of elasticity, arterial insufficiency and retinal haemorrhages leading to macular degeneration. PXE is usually found as a sporadic disorder, but examples of both autosomal recessive and autosomal dominant forms of PXE have been observed. Partial manifestations of the PXE phenotype have also been described in presumed carriers in PXE families. Linkage of both dominant and recessive forms of PXE to a 5-cM domain on chromosome 16p13.1 has been reported (refs 8,9). We have refined this locus to an 820-kb region containing 6 candidate genes. Here we report the exclusion of five of these genes and the identification of the first mutations responsible for the development of PXE in a gene encoding a protein associated with multidrug resistance (ABCC6).