ectopic mineralization of soft connective tissues , particularly the cardiovascular system , is globally a major cause of morbidity and early mortality ( budoff et al . , 2007 ) .
the pathomechanisms leading to deposition of mineral complexes in these tissues are exceedingly complex , but significant insights have been gained from observations in a group of heritable ectopic mineralization disorders with defined gene defects ( li and uitto , 2013 ) .
the prototype of such conditions is pseudoxanthoma elasticum ( pxe ) , a multi - system disorder characterized by deposition of calcium hydroxyapatite in various connective tissues , with protean manifestations ( neldner , 1988 ; uitto et al . , 2013 ) .
the classic form of pxe is of late - onset and slowly progressing , and the major clinical problems relate to loss of eyesight and development of vascular complications ( neldner , 1988 ) .
another , characteristically more severe ectopic mineralization disorder is the generalized arterial calcification of infancy ( gaci ) , frequently diagnosed by prenatal ultrasound revealing vascular calcium deposits ( nitschke and rutsch , 2012a ; rutsch et al . , 2011 ) .
the newborns manifest with cardiovascular complications and many of these patients die within the first year of life , particularly if not receiving bisphosphonate treatment ( see below ) .
finally , a more recently described condition , arterial calcification due to cd73 deficiency ( acdc ) , manifests with calcification of arterial blood vessels particularly in the lower extremities and in periarticular ligaments and tendons , typically encountered in older individuals ( markello et al .
thus , these clinical entities all manifest with varying degrees of mineralization of the arterial blood vessels , with evidence of phenotypic overlap ( le boulanger et al . , 2010 ; li et al .
significant progress has recently been made in understanding the genetic basis of these heritable ectopic mineralization disorders ( table 1 ) .
specifically , the classic form of pxe is now known to be caused by loss - of - function mutations in the abcc6 gene encoding atp - binding cassette subfamily c , member 6 ( abcc6 ) , a putative transmembrane efflux transporter protein expressed primarily in the baso - lateral plasma membrane of hepatocytes and in the proximal tubules of the kidney ( belinsky and kruh , 1999 ; pomozi et al . , 2014 ; scheffer et al . , 2002 ; uitto et al . ,
the molecules transported physiologically by abcc6 from the intracellular milieu to extracellular space have not been identified as yet , but recent studies have demonstrated that abcc6 is required for the release of atp from the hepatocytes raising the question whether atp , is the physiologic target molecule to be transported by abcc6 ( jansen et al . , 2014 ; jansen et al . , 2013 ) .
pxe is characterized by considerable both intra- and inter - familial heterogeneity , suggesting a role for genetic and environmental modifying factors .
researchers are now taking candidate gene and whole exome sequencing approaches to further dissect out the effects of putative modifier genes on clinical presentations in pxe ( dabisch - ruthe et al .
in addition , there are reports that alterations in lipid metabolism and soft tissue calcification are closely related pathologies , and in this context , the role of abcc6 deficiency has been studied in lipoprotein and cholesterol homeostasis and in the development of atherosclerosis ( kuzaj et al . , 2014 ;
several studies performed on pxe fibroblasts have demonstrated that mesenchymal cells , whether locally producing pro- and anti - calcifying factors or being involved in extracellular matrix synthesis and degradation , are involved in the mineralization of soft connective tissues ( boraldi et al . , 2014 ; ronchetti et al . , 2013
) . however , it is unclear at the present if all mesenchymal cells in pxe lesions behave similarly , or whether their tissue - specific differentiation contributes to different susceptibility of connective tissues to mineralization .
the classic form of gaci is caused by mutations in the enpp1 gene which encodes ectonucleotide pyrophosphatase / phosphodiesterase 1 ( enpp1 ) , an enzyme facilitating hydrolysis of atp to amp and inorganic pyrophosphate ( ppi ) ( fig .
1 ) . ppi is a powerful anti - mineralization factor , while inorganic phosphate ( pi ) serves as a pro - mineralization factor , and consequently , an appropriate ratio of ppi / pi is required to prevent spontaneous calcium phosphate precipitation under normal homeostasis . in the absence of enpp1 , this ratio is reduced and ectopic mineralization ensues .
interestingly , recent studies have demonstrated that some patients with pxe - like clinical features harbor mutations in the enpp1 gene while some patients clinically diagnosed as gaci have mutations in the abcc6 gene ( li et al .
therefore , these genotypic findings can explain the overlapping phenotypic features in some patients with ectopic mineralization disorders , pointing to shared pathomechanistic pathways leading to ectopic mineralization .
patients with acdc with vascular calcification have been shown to harbor mutations in the nt5e gene which encodes cd73 , a membrane - bound ecto-5-nucleotidase that breaks down amp to adenosine and pi ( markello et al . , 2011 ; st hilaire et al . ,
cd73 deficiency also increases the activity of tissue non - specific alkaline phosphatase ( tnap ) , accelerating conversion of ppi to pi .
adenosine is a physiologic inhibitor of tnap , and reduced adenosine concentration results in tnap activation further facilitating the conversion of ppi to pi ( fig .
in patients with pxe , the mutation database consists of ~300 distinct loss - of - function mutations in the abcc6 gene , including recurrent p.r1141x and g.del2329 mutations , which account for up to ~45% of all reported pathogenic alleles ( terry and hefferson , 2013 ) .
it should be noted , however , that most of the findings have been reported in caucasian patients from the united states and europe , and for example , only recently , mutations in pxe patients of asian ancestry have appeared in press .
specifically , a recent study on chinese patients with pxe revealed mostly previously unreported mutations , and there is an apparent paucity of the recurrent p.r1141x and g.del2329 mutations ( jin et al . , 2015 ) .
this observation emphasizes the importance of studying patients of various ethnic backgrounds and different ancestries to allow development of allele - specific therapies for pxe .
significant progress in understanding of the pathomechanistic details leading to ectopic mineralization in these disorders has been made by examination of animal models , particularly mice ( nitschke and rutsch , 2012b ) .
specifically , histopathologic examination of abcc6 knock - out mice ( abcc6 and abcc6 ) demonstrated the presence of mineral deposits in a number of tissues , including skin , eyes , and the cardiovascular system , similar to that in patients with pxe ( gorgels et al .
in addition to genetically engineered knock - out mice , a number of inbred mouse strains , such as kk / hlj , have been demonstrated to harbor a specific mutation in the abcc6 gene which predisposes these animals to ectopic mineralization similar to the corresponding targeted knock - out mice ( berndt et al . , 2013 ; li et al . , 2012a ) .
it should be noted that the degree of mineralization in these different mouse strains is highly variable even under standardized laboratory conditions , attesting to the presence of putative modifier genes ( li et al . , 2013a ) .
in addition to the mouse models , a rat model through inactivation of abcc6 gene using zinc finger nuclease technology has recently been developed .
preliminary data demonstrated mineralization of the dermal sheath of vibrissae of the homozygous mutant rat , similar to findings in abcc6 knock - out mice ( li et al . ,
unpublished ) . another animal model for pxe consists of a zebrafish which has an orthologous abcc6a gene .
knock - down of the abcc6a gene in 14 cell stage embryos by a morpholino , a stable antisense nucleotide eliciting suppression of expression of the targeted gene , results in profound phenotype consisting of pericardiac edema , stunted and curved tail ( li et al . , 2010 ) .
this phenotype can be readily rescued by concomitant injection of human or mouse abcc6 mrna together with the morpholino , attesting to the specificity of the abcc6a phenotype .
this model has provided a system to assess the pathogenicity of missense mutations in the human abcc6 gene by testing whether such mutant mrnas do or do not rescue the phenotype ( jin et al .
the missense mutation is deemed to be pathogenic , especially when combined with bioinformatics analysis .
in addition to the morpholino - mediated abcc6a knock - down system , a zebrafish grt mutant was recently identified in a forward genetic screen , with a p.l1429r mutation in the abcc6a gene ( mackay et al . , 2015 ) .
in contrast to morpholino - injected embryos with early lethality , the grt mutant fish survived for at least one year .
both heterozygous and homozygous mutant fish show signs of excessive mineralization in the craniofacial region and axial skeleton , and infrequently also in the skin .
in addition , abcc6a was found to be strongly expressed at the sites of mineralization rather than in the liver , as it is in human and mice .
therefore , the grt phenotype does not fully recapitulate that of humans with pxe due to bi - allelic mutations in the abcc6 gene .
a number of animal models , particularly targeted and spontaneous mutant mice , have also been identified and characterized to recapitulate the features of gaci caused by enpp1 mutations .
 tip toe walking ( enpp1/enpp1 ) was first described as a spontaneous mutant mouse harboring a p.g568x nonsense mutation in the enpp1 gene ( okawa et al . , 1998 ) .
these mice exhibit accelerated bone formation , ossification of the spinal ligaments and ectopic mineralization of the aortic medial layer at the level of the internal elastic lamina .
enpp1 knock - out mice exhibit abnormalities similar to those in  tip toe walking  mice ( sali et al . , 1999 ) . another enpp1 mutant mouse ( enpp1 ) , with a p.c397s missense mutation , has been characterized by low bone mineral density , crystal - related arthropathy and vascular calcification ( babij et al . , 2009 ) . a spontaneous mutant mouse with similar phenotype ( enpp1 )
these mice were shown to harbor a splice - site mutation c.259 + 1g > t in enpp1 gene ( takabayashi et al . , 2014 ) .
a mutant mouse with a missense mutation ( p.v246d ) in the enpp1 gene was recently identified at the jackson laboratory as a result of enu treatment ( li et al . , 2013b ) .
these mice demonstrated stiff posture , abnormalities in the front legs and stiffening of the joints ; hence , this mutation was named 
the lack of enpp1 enzymatic activity in asj homozygous mice results in reduced ppi levels in the plasma , accompanied by extensive mineralization of a number of tissues , including arterial blood vessels and the dermal sheath of vibrissae .
another mutant mouse , asj-2j , was also identified at the jackson laboratory with a phenotypic gait due to periarticular mineral deposits .
this mouse was noted by histology to have extensive mineralization of the dermal sheath of vibrissae as well as arterial blood vessels .
the asj-2j mice were shown to be allelic to asj mice by complementation studies , and these mice harbor of a large 40,035 bp deletion spanning from intron 1 to the 3-untranslated region of the enpp1 gene , coupled with a 74 bp insertion , thus completely eliminating functional gene in these mice ( li et al . , 2014a ) .
thus , these targeted and spontaneous mutant mice display features of gaci , and can serve as a platform to explore treatment of this ectopic mineralization disorder .
this mouse ( nt5e ) , developed by ablation of the nt5e gene ( castrop et al . , 2004 ) , showed stiffening of the joints , and micro ct revealed distinct changes in the thoracic skeletal structure with profound mineralization of the costochondral junctions ( li et al . , 2014b ) .
mineralization was also noted in juxta - articular joint - capsules as well as ligaments adjacent to the bony structures , features in patients with acdc .
no evidence of vascular mineralization was noted , indicating that the nt5e knock - out mice recapitulate some , but not all , features of acdc . there was a markedly reduced ppi / pi ratio in the plasma , attesting to the presence of a complex promineralization/ anti - mineralization network , characteristic to ectopic mineralization disorders , particularly in this case in acdc .
the metabolic hypothesis concerning pxe postulates that the absence of functional abcc6 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 ( jiang and uitto , 2006 ) .
however , the physiological role of abcc6 protein is currently unknown , and in particular , the identity of the factor(s ) being transported in vivo has long remained a mystery .
recently , using cultured cells , abcc6 was found to mediate the cellular release of atp which is rapidly converted extracellularly into amp and ppi ( jansen et al . , 2014 ; jansen et al . , 2013 ) .
outside hepatocytes , but still within the liver vasculature , released atp is converted into amp and ppi , revealing an unanticipated role of the liver in systemic ppi homeostasis .
these findings raised the question whether atp is the physiologic substrate of abcc6 , however , transport of atp outside of the cells has not been demonstrated so far .
abcc6 , enpp1 , and nt5e , respectively , are all part of the pathway that involves generation of ppi and pi from atp ( fig .
1 ) . the unifying feature in these patients is reduction in the plasma ppi concentration resulting in reduced ppi / pi ratio .
since ppi is a powerful anti - mineralization factor and the primary deficiency in enpp1 or cd73 activity results in depletion of the ppi pool , it appears that these two conditions are pathomechanistically related .
furthermore , in the absence of functional abcc6 , the release of atp to the extracellular milieu is reduced , potentially lowering the extracellular pool of atp .
in such situation , less atp is available as a substrate for enpp1 , again resulting in reduced ppi plasma concentration ( fig .
1 ) . in support of this hypothesis are recent demonstrations that ppi levels in the plasma of patients with pxe are reduced leading to lower ppi / pi ratio ( jansen et al . , 2014 ; jansen et al . ,
thus , all three ectopic mineralization disorders , pxe , gaci and acdc , with overlapping phenotypic features , share the deficiency in the same metabolic pathway .
the phenotypic differences in these conditions may reflect the specific additional roles of the mutated genes when combined with modifying genetic factors and the environmental influences .
nevertheless , understanding the pathomechanistic alterations in these diseases may provide targets for pharmacologic modulation of the disease .
no specific treatment modality is currently available for prevention of systemic ectopic mineralization in pxe or gaci .
remarkable success has been achieved , however , in the treatment of ocular complications in pxe using vascular endothelial growth factor antagonists ( lucentis and avastin ) which prevent neovascularization and preserve the vision ( myung et al . , 2010 ; verbraak , 2010 ) .
in fact , this approach has markedly reduced the incidence of the most severe complication of pxe , i.e. , loss of vision , and has changed the focus of the disease to be largely on the skin and arterial vasculature . the first attempts to develop treatment for systemic mineralization in pxe focused on modifications of the mineral content of the diet .
preclinical animal studies , using abcc6 knock - out mice as a platform , demonstrated that a diet with elevated phosphorus and reduced magnesium content ( so - called  acceleration diet  ) enhanced the ectopic mineralization in this model , while conversely , increasing the magnesium content in the mouse diet by 5-fold completely abolished ectopic mineralization ( jiang and uitto , 2012 ; larusso et al . , 2009 ) .
furthermore , recent studies have demonstrated that feeding of pregnant mice with the  acceleration diet  increases the mineralization of the newborn pups , with apparent relevance to gaci characterized by ectopic mineralization of blood vessels during fetal development ( li et al . , 2015b ) .
based on the observations of the critical role of magnesium in modifying the degree of mineralization , a double - blinded placebo - controlled clinical trial with diet fortified with magnesium has been initiated in patients with pxe ( https://clinicaltrials.gov/ct2/show/nct01525875 ) .
considering the evidence in support of the notion that reduced ppi concentration is a critical factor allowing ectopic mineralization to ensue , a suggestion has been made that introduction of ppi would be helpful in prevention of the mineralization processes ( o'neill et al .
unfortunately , inorganic pyrophosphate is extremely labile and has a short half - life due to hydrolysis of the molecule .
this has raised the possibility to use stable , non - hydrolysable pyrophosphate analogs , bisphosphonates , for treatment of ectopic mineralization disorders .
in fact , bisphosphonates have been used for either prenatal or perinatal treatment of gaci patients , but careful examination of the literature reveals varying results , some studies showing apparent improvement , while in others very little , if any , effect is found ( edouard et al . , 2011 ; galletti et al . , 2011 ;
furthermore , bisphosphonate administration has resulted in severe toxicity after protracted therapy ( otero et al . ,
2013 ) . to explore the efficacy of bisphosphonates for treatment of ectopic mineralization disorders , recent studies have used abcc6 knock - out and asj mice as a model of pxe and gaci , respectively ( li et al .
in principle , bisphosphonates have two properties on mineralization : ( i ) anti - mineralization activity which is derived from incorporation of the bisphosphonate into calcium phosphate crystals preventing their growth , and ( ii ) anti - osteoclastic activity which prevents bone resorption .
the latter activity is the basis for extensive use of bisphosphonates for treatment of osteoporosis and other disorders affecting bone , including paget s disease , bone metastases , osteogenesis imperfecta and multiple myeloma ( uludag , 2002 ) .
bisphosphonates , etidronate or alendronate , demonstrated that they were able to prevent ectopic mineralization in abcc6 or asj mice when administered either by mouth at relatively high concentrations or by subcutaneous administration in 100-times lower concentration ( li et al .
interestingly , the microarchitecture of the bones , which is severely perturbed in the asj mice , was corrected with etidronate .
thus , there is a dual beneficial effect , i.e. , inhibition of soft tissue mineralization and improvement in bone microarchitecture , which was achieved by bisphosphonate administration .
it should be noted , that these beneficial effects were achieved with relatively high concentrations of bisphosphonates , and further long - term toxicity studies are required before this approach should be tested in clinical trials in humans , particularly concerning potential side effects .
the potential treatment of pxe has also recently been explored by a number of molecular and cell - based strategies .
for example , the potential correction of nonsense mutations in the abcc6 gene by read - through mechanism facilitated by ptc124 , a non - aminoglycoside nonsense mutation suppressor , has been tested ( zhou et al . , 2013 ) .
preliminary studies demonstrated that ptc124 enhances read - through of stop codon mutations in this gene in cell culture systems .
with respect to missense mutations with preserved transport activity but with improper plasma membrane localization of the protein , a chemical chaperone , 4-phenylbutyrate ( 4-pba ) , a drug approved for clinical use for other indications , has been tested in pxe mice .
the results revealed restoration of the plasma membrane localization of abcc6 in mouse hepatocytes in a limited number of specific mutations , suggesting that allele - specific therapy may be useful for selected patients with pxe and gaci , i.e. , those carrying missense mutations in their abcc6 gene ( le saux et al .
furthermore , transplantation of cells with hepatoblastic lineage differentiation , either representing a hepatoblast cell line or differentiated from induced pluripotent stem cells ( ipscs ) , has demonstrated homing of these cells to the liver and has resulted in abcc6 protein expression in abcc6 knock - out mice ( jiang et al .
significant progress has been made in understanding the molecular basis and pathomechanistic pathways involved in heritable ectopic mineralization disorders , as exemplified by pxe , gaci and acdc .
this progress has now identified pathways that can potentially serve as pharmacologic targets towards treatment of these currently intractable disorders .
while these heritable disorders are rare , study of these conditions has allowed significant insights into complex , much more common disorders .
for example , utilization of mouse models of pxe , the level of hepatic expression of abcc6 has been shown to determine the severity of calcification and the infarct size after cardiac injury , contribute to arterial calcification , and participate in development of chronic kidney disease ( brampton et al . , 2014 ;
lau et al . , 2014 ; mungrue et al . , 2011 ; prunier et al
in that sense , the abcc6 transporter system can be considered as a paradigm for extending the knowledge from rare heritable diseases to complex disorders ( de vilder et al . , 2015 ) . finally ,
development of novel pharmacologic interventions for rare heritable ectopic mineralization disorders , such as pxe , may be applicable to disorders , such as arteriosclerosis , a much more common condition in general populations , with a major burden to the global healthcare systems .
 heritable ectopic mineralization disorders represent a phenotypically diverse group of conditions characterized by deposition of calcium phosphate complexes in soft connective tissues . 
 the prototype of such conditions is pseudoxanthoma elasticum ( pxe ) , and related conditions with overlapping clinical features include generalized arterial calcification of infancy ( gaci ) and arterial calcification due to cd73 deficiency ( acdc ) . 
 molecular genetic investigations have revealed mutations in the genes physiologically involved in generation of inorganic pyrophosphate ( ppi ) and phosphate ( pi ) , and the findings suggest a unifying pathomechanism relating to reduced ppi / pi ratio . 
 this hypothesis is based on the notion that ppi serves as a powerful inhibitor of mineralization while pi is a pro - mineralization factor , and an appropriate ppi / pi ratio is critical for prevention of ectopic mineralization under homeostatic conditions.pxe international , the premiere patient support organization , advocating on behalf of patients and families with pxe , sponsors regular research meetings evaluating the progress in this and related ectopic mineralization disorders . the latest meetings were held in september 2014 in bethesda , md and in september 2015 in budapest , hungary . 
 this report summarizes the latest progress in research on pxe and related ectopic mineralization disorders , based on presentations and discussions in these meetings , with pharmacologic implications for currently intractable disorders . 
Introduction
Genetic Bases and Phenotypic Modulation
Molecular Diagnostics
Animal Models for Heritable Ectopic Mineralization Disorders
The Unifying Concept of Pathomechanistic Pathways
Development of Novel Treatment Modalities
Conclusions