Pseudoxanthoma Elasticum, the Paradigm of Heritable Ectopic Mineralization Disorders - Can Diet Help?

SUMMARY

Pseudoxanthoma elasticum (PXE) is a heritable multi-system disorder manifesting with characteristic cutaneous lesions, associated with ocular findings and cardiovascular involvement. The skin lesions, yellowish papules which coalesce into plaques of inelastic and leathery skin, demonstrate by histopathologic and ultrastructural examinations ectopic mineralization of dermal connective tissues, primarily the elastic structures. PXE is inherited in an autosomal recessive fashion due to mutations in the ABCC6 gene. Significant insights into the pathogenesis of PXE have been recently obtained from observations on the Abcc6^−/− knockout mouse which mimics the genetic, histopathologic and ultrastructural features of PXE. This mouse model has provided a platform to test various treatment modalities to counteract the mineralization phenotypes. One of the intriguing findings emanating from these studies is that supplementation of the mouse diet with magnesium, at levels that are ~5-fold higher than those in control diet, completely inhibits the development of tissue mineralization. These and related observations suggest that changes in the diet might counteract the progression of PXE and improve the quality of life of patients with this, currently intractable, disease.

The Clinical Spectrum of PXE

Pseudoxanthoma elasticum (PXE), the prototype of heritable multi-system disorders characterized by ectopic mineralization of connective tissues, manifests with clinical involvement of the skin, the eyes, and the cardiovascular system (Table 1). The precise prevalence of PXE is currently unknown, but it is estimated to be around 1:50,000, with a carrier frequency of ~1:150-300. PXE is caused by mutations in the ABCC6 gene which encodes ABCC6, a transmembrane transporter protein [for review, see ref. 1, 2].

Table 1. Clinical Features of Pseudoxanthoma Elasticum.

Skin Findings

Yellowish papules on the predilection sites in the skin and mucous membranes Lax and inelastic skin Pleiomorphic elastic structures in the dermis with progressive calcification

Eye Findings

Angioid streaks (very diagnostic); peau d’orange; comet lesions (rare); scarring (late) Retinal neovascularization with loss of visual acuity Legal blindness (rare)

Cardiovascular Findings

Calcification of arterial blood vessels Hypertension, intermittent claudication, absent peripheral pulses Bleeding from internal arteries (rare) Early myocardial infarcts (rare)

Genetics

Family history with autosomal recessive inheritance Mutations in the ABCC6 gene

PXE can pose a diagnostic challenge to practicing dermatologist for several reasons. First, although it is a fully penetrant autosomal recessive disorder, clinical manifestations of PXE are rarely present at birth, and skin findings usually become recognizable not until the second or third decade of life. In many cases, accurate diagnosis is not made until after several years of delay when serious ocular or vascular complications develop. Secondly, there is considerable both intra- and inter-familial heterogeneity, so that in some families the skin manifestations may be predominant with relatively little eye or cardiovascular involvement, while in other families involvement of the latter organ systems may have severe clinical consequences with considerable morbidity and occasional early mortality [1]. The reasons for this phenotypic heterogeneity are currently unclear, and attempts to establish genotype/phenotype correlations in different populations have yielded largely negative results. There are suggestions, however, that the contribution of genetic modifier genes, epigenetics, dietary factors, and life style variables contribute to the variable phenotypes [2].

Adding to the clinical diagnostic difficulties relating to PXE are the observations that cutaneous manifestations akin to those in PXE can be encountered in a number of both acquired and heritable, genetically unrelated clinical conditions (Table 2). For example, PXE-like cutaneous changes can be encountered in patients with β-thalassemia and sickle cell anemia [3, 4]. A particularly intriguing observation, with potential mechanistic implications for PXE, is that PXE-like cutaneous findings can be noted in association with vitamin K-dependent multiple coagulation factor deficiency [5-7]. Some of these patients show lesions similar to those seen in PXE, although in many of these patients there is also an excessive folding and sagging of the skin similar to cutis laxa (Figure 1). However, skin biopsy from these patients depicts characteristic mineralization of elastotic material that accumulates in the mid-dermis, similar to PXE, a finding not present in patients with cutis laxa.

Table 2. Differential Diagnosis of Pseudoxanthoma Elasticum.

Skin Findings (with mineralization of elastic fibers)

PXE-like cutaneous changes due to mutations in the GGCX gene Skin findings in β-thalassemia and sickle cell anemia Acquired PXE due to topical exposure to calcium/phosphate salts

Skin Findings (without mineralization)

Solar elastosis Papillary dermal elastolysis and mid-dermal elastolysis Buschke-Ollendorff syndrome and isolated elastomas Elastofibroma dorsiD-penicillamine treatment (long-term)

Eye Findings

Age-associated macular degeneration

Cardiovascular Mineralization

Generalized arteriosclerosis Idiopathic infantile arterial calcification (EPPP1 mutations)

Figure 1.

Clinical manifestations and histopathology of skin in patients with PXE in a family with mutations in the ABCC6 and GGCX genes. (a) In a patient compound heterozygous with mutations in one allele of each of the genes shows cutaneous findings characteristic of PXE, including small yellowish papules in the axillary area (arrow). (b) The 18-year old proband, who is compound heterozygous for missense mutations in the GGCX gene, demonstrates, in addition to yellowish papules characteristic of PXE, redundant, loose and sagging skin. Histopathology of skin by special stains (c, von Kossa stain; d, Alizarin red) shows characteristic mineralization in the mid-dermis. (Modified from reference 7).

Finally, while recent molecular studies based on mutation detection have clearly indicated that PXE is an autosomal recessive disorder, a few cases have been suggested to demonstrate clinical findings in patients with a mutation in only one ABCC6 allele [8, 9]. Careful examination of these cases reveals the presence of subtle findings, and in some cases the family relationships are not entirely clear. Furthermore, several studies on relatively large PXE cohorts have failed to identify clinical findings suggestive of PXE in heterozygous carriers [10-12]. Thus, as a general rule, the overwhelming evidence suggest that virtually all families with PXE display autosomal recessive inheritance. This situation then allows appropriate counseling for the genetic risk of inheritance of an affected child either in the same or in subsequent generations and allows presymptomatic testing of siblings with affected individuals in the family [2, 13].

Molecular Genetics

The histopathology of the skin in PXE was initially noted to involve mineralization of elastic fibers, and consequently, the genes participating in the synthesis and assembly of the elastic fiber network were considered as candidate gene/protein systems for mutations in this disease; such genes included elastin (ELN), fibrillins (FIB1, 2), fibulins (FBN2, 4, 5) and lysyl oxidase (LOX). However, with the advent of cloning of the corresponding genes, genetic linkage analyses systematically excluded these genes and the corresponding chromosomal regions as the site of PXE associated genes. Subsequently, with the support of PXE International, a patient advocacy organization actively partnering with scientists on PXE research [14, 15], positional cloning studies were commenced which provided evidence for linkage to the short arm of chromosome 16 [refs. 16, 17]. Refinement of the critical region finally pinpointed ABCC6 gene as the one harboring mutations in PXE [18-21]. This gene encodes a putative transmembrane transporter protein, ABCC6, which has been postulated to function as an efflux transporter, however, the precise role of ABCC6 and the substrate specificity of this putative transporter in vivo are currently unknown.

Well over 300 distinct mutations representing over 1,000 mutant alleles have been encountered in the ABCC6 gene in patients with PXE [10-12, 22]. The spectrum of mutations include missense and nonsense mutations, splice-junction mutations resulting in mis-splicing, small deletions and insertions resulting in in-frame shift of translation, as well as large deletions spanning part of the entire coding region of the ABCC6 gene and in some cases flanking genes as well. Collectively, mutation analysis is now readily available for individuals suspected to have or to be at risk for PXE. Identification of specific mutations in the ABCC6 gene can now be used for confirmation of the clinical diagnosis, carrier detection, and presymptomatic identification of affected individuals. Consequently, early identification of the disease and increased surveillance for its sequela will undoubtedly improve the quality of life of the affected individuals.

Mouse Models

Soon after the first demonstration of mutations in the ABCC6 gene in humans with PXE, transgenic mouse models were developed through targeted ablation (knock-out) of the Abcc6 gene [23, 24]. These Abcc6^−/− mice recapitulate features of human PXE, including autosomal recessive inheritance with full penetrance, and delayed onset of mineralization which is evident by histopathologic examination first around 5-6 weeks of age. Similar to findings in patients with PXE, the Abcc6^−/− mice demonstrate deposition of mineral complexes in the skin, the retina and the arterial blood vessels, in addition to widespread evidence of mineralization in other tissues as well (Figure 2). A characteristic feature of Abcc6^−/− mice is the early and progressive mineralization of the connective tissue capsule surrounding the vibrissae [24]. Subsequently, assessment of the degree of mineralization of vibrissae by histopathology, coupled with computerized morphometric analysis, and by direct chemical assay of calcium and phosphate in tissues, can serve as a reliable and quantitative biomarker to follow the progression of mineralization. Thus, the Abcc6^−/− mice have provided a useful preclinical model system to explore the pathomechanisms of PXE and to test potential treatment modalities [25 26].

Figure 2.

Schematic representation of the putative pathomechanisms of PXE. Under physiological conditions, ABCC6, a putative transmembrane transporter protein expressed primarily in the liver, removes currently unidentified molecules, which potentially serve as anti-mineralization factors, from hepatocytes to circulation. In the absence of ABCC6 activity, the substrate molecules accumulate in the liver, and reduced anti-mineralization activity in the circulation and peripheral tissues allows the mineralization processes to ensue in the eyes, blood vessels, kidneys and skin, as depicted by Alizarin red staining of the corresponding tissues in Abcc6^−/− mice. (Reproduced from reference 2).

Towards Treatment of PXE

Histopathologic evaluation of skin in patients with PXE reveals progressive mineralization of connective tissues, and the elastic structures in particular, as a characteristic feature [1]. Accumulation of calcium/phosphate complexes in these lesions is apparently responsible for clinical manifestations in the skin, in the eyes, and the cardiovascular system. The ABCC6 gene harboring mutations in PXE is expressed primarily in the liver, to a lesser extent in the kidneys and the intestine, and at very low level, if at all, in tissues clinically affected with PXE [27, 28]. This and various experimental observations in animal models of PXE have suggested that PXE is a metabolic disorder, postulating that absence of functional ABCC6 transporter activity primarily in the liver results in deficiency of circulating factor(s) that are physiologically required to prevent aberrant mineralization under normal calcium and phosphate homeostatic conditions [2, 29](Figure 2). In this context, it is important to emphasize that no abnormalities in the serum calcium or phosphate content have been noted, and parathyroid hormone levels are within the normal limits.

Previous studies have identified a number of proteins that can act as powerful anti-mineralization factors in the circulation, including matrix Gla-protein (MGP), which needs to be activated by γ-glutamyl carboxylase, a vitamin K-dependent enzyme, to restore the full anti-mineralization capacity of this molecule [30, 31]. This observation, together with the findings that mutations in the GGCX gene, encoding the γ-glutamyl carboxylase, can cause PXE-like cutaneous manifestations, suggested that PXE may result from reduced concentrations of vitamin K or its derivatives in peripheral tissues, resulting in reduced activation of anti-mineralization proteins [5-7, 32]. This hypothesis has been further supported by the notion that the ratio of active (carboxylated) compared to inactive (under-carboxylated) forms of MGP has been shown to be reduced in tissues of patients with PXE and of Abcc6^−/− mice [33, 34]. Collectively, these findings suggest that vitamin K deficiency may play a role in pathogenesis of PXE and that administration of vitamin K may counteract the mineralization process [2]. This hypothesis is currently being tested in different model systems of PXE.

A key challenge for development of effective and specific treatment modalities for PXE resides in identification of the transport substrate(s) for ABCC6. At the same time, it is recognized that clinical manifestations in PXE are primarily, if not exclusively, related to ectopic mineralization of peripheral tissues. Thus, anti-mineralization approaches aimed at prevention of calcium/phosphate deposition in tissues might provide a treatment for PXE.

The Role of Diet

Recent studies have focused on the role of the mineral content of diet in modifying the severity of PXE. These studies were originally based on retrospective surveys which suggested that individuals with a history of high intake of dairy products rich in calcium and phosphate during adolescence developed more severe disease later in life [1, 35]. Recently, genetically controlled studies utilizing the Abcc6^−/− mouse as the preclinical platform have specifically shown that magnesium, but not calcium, content of the diet can influence the extent of ectopic mineralization in peripheral tissues [36-39]. Specifically, supplementation of the mouse diet with magnesium carbonate in amounts that increase the magnesium content by 5-fold over the standard diet, completely abolished the mineralization noted in the Abcc6^−/− mice [37, 38]. Conversely, the experimental diet with low magnesium was shown to accelerate the mineralization process [40]. While the mechanisms by which magnesium prevents calcium/phosphate deposition in tissues in this animal model are currently unknown, there was a marked increase in the urinary output of calcium with concomitant reduction in phosphate in the magnesium treated mice. It is possible, therefore, that magnesium replaces calcium in the calcium/phosphate complexes, and since magnesium phosphate is more soluble than calcium phosphate in the corresponding concentrations, this may result in reduced or absent mineralization in tissues. It should be noted that the calcium content of the bones measured by chemical assay and bone density determined by micro computerized tomography scan did not show any long term adverse effects on the bones as a result of extended increase of magnesium intake in the Abcc6^−/− mice [37]. Thus, these findings support the notion that changes in the diet, specifically changes in dietary magnesium, might be helpful for patients with PXE [2]. The dietary allowance of magnesium recommended by the Food and Drug Administration (U.S.) is up to 420 mg. per day for adults [41], but doubling this dose would expected to have minimal side effect. In this context, it should be noted that various vegetables (spinach, tomato and pumpkin seeds) and grains (buckwheat flour, oat bran, barley and wheat flour) are particularly rich in magnesium [41]. Based on these preclinical mouse studies, a clinical trial exploring the effects of supplementary dietary magnesium on the progression of cutaneous and ocular signs in PXE is currently being developed (Mark Lebwohl, Personal Communication). One difficulty of such clinical trials in humans is that the clinical course of PXE is slow and quite variable, and there are no reliable biomarkers to predict the improvement short-term. Finally, preliminary studies on oral phosphate binders have suggested an improvement in the degree of skin manifestation of patients with PXE, and these studies are being extended to larger cohorts of patients [42, 43].

Future Perspectives

There are a number of hurdles/obstacles that may hamper efficient design of clinical trials for PXE. A major impediment is the lack of knowledge of the precise pathomechanisms leading from the mutations in the ABCC6 gene to the mineralization of the peripheral connective tissues. Furthermore, the phenotypic heterogeneity as well as slow and unpredictable progression of the clinical involvement in PXE in individual patients makes documentation of the effectiveness of a test compound difficult and extends the time period required for determination of an unequivocal improvement in the end points. This difficulty is emphasized further by the fact that currently there are no established or validated biomarkers for PXE which would allow earlier determination of the efficacy of the treatment modalities. At the same time, one could envision a number of molecular strategies, many of which could be adapted from the technical advances made in other fields. Some of these approaches could take advantage of the knowledge of specific mutations identified in the ABCC6 gene in PXE. For example, development of drugs that facilitate read-through of premature termination codon-causing mutations, so as to allow synthesis of the full-length protein from the mutant allele, would be potentially beneficial for many patients with PXE [44].

Another mutation-specific approach would involve assisted trafficking of mis-targeted and mis-folded proteins by utilizing chaperones, modifiers of protein conformation, and transporter activators, approaches that have been developed for heritable diseases caused by mutations in other ABC transporter genes, such as cystic fibrosis [45, 46]. Another approach, dependent of the specific types of mutations in ABCC6, could target splice-junction mutations which result in miss-splicing of pre-mRNA. However, some of these splice-site mutations are “leaky” allowing low level of baseline expression of functional, fully spliced protein. In such case, up-regulation of the corresponding mutant allele by cytokines, specific transcription factors, or small molecular weight compounds that can be screened in large scale fluorescence-based assay systems, might be beneficial [2]. Finally, cell-based therapies taking advantage of allogeneic stem cells derived from bone marrow or umbilical cord, or development of inducible pluripotent stem cells with potential to differentiate into hepatocytic lineage in the liver, might provide an approach for regenerative processes [47]. Also, development of patient-specific inducible pluripotent stem cells, coupled with correction of the underlying molecular defect, could lead to personalize regenerative medicine for treatment of PXE. However, before these molecular approaches are ready for translation into clinics, a combination of empiric therapies, including appropriate diet, which may include supplementations with magnesium, vitamin K or phosphate binders, either individually or in combination, might slow down the progression of PXE and improve the quality of life of patients with this, currently intractable, disease.