Medial arterial calcification of the lower extremities with periarticular calcification was described first by Magnus-Levy15
in 1914 and again by Levitin16
in 1945. The familial nature of this condition was first suggested in a report on two affected siblings by Sharp17
in 1954, leading to a subsequent record in the Online Mendelian Inheritance in Man database (OMIM number, 211800). Other, single cases were described by Nosaka and colleagues18
and Mori and coworkers,19
yielding a total of seven cases published to date. Here, we describe the molecular and enzymatic basis of this disorder in nine patients with three different mutations in NT5E
Considerable evidence supports the association of these families’ vascular disease with mutations in the NT5E gene. The results of segregation analysis were consistent among our families, and the nonsense mutation (p.S221X) and single-nucleotide insertion (p.V537fsX7) predict truncated CD73 proteins. The missense mutation (p.C358Y), which was not found in 200 unaffected persons, predicts a pathologic change in an amino acid conserved through evolution and is located in the critical nucleotidase domain of CD73. Furthermore, the nonsense mutation resulted in markedly reduced levels of CD73 mRNA and protein in cultured cells. Enzyme activity was virtually absent in fibroblasts from affected members of Family 1 and was rescued by transduction of a lentiviral vector expressing NT5E. Each of the three different mutations in the three families produced essentially nonfunctional CD73.
CD73 participates in the extracellular pathway that converts ATP to adenosine on the surface of various types of cells, as follows. First, ENPP1 produces AMP and pyrophosphate from ATP; then CD73 produces adenosine and inorganic phosphate from AMP (). Cellular calcification depends critically on levels of pyrophosphate, a strong inhibitor of calcification, and TNAP, which degrades pyrophosphate.14
In patients with hypophosphatasia due to TNAP deficiency, increased levels of pyrophosphate result in defective bone mineralization.20
In patients with generalized arterial calcification of infancy, ENPP1 deficiency leads directly to decreased pyrophosphate levels,21
causing early-onset vascular calcification, myocardial infarction, and often death in infancy.5
In our adult patients, CD73 deficiency may not lead directly to decreased pyrophosphate levels, but the consequent reduction in extracellular adenosine levels apparently enhances TNAP activity; adenosine supplementation reversed the increase in TNAP activity in CD73-deficient cells. We hypothesize that increased TNAP activity reduces pyrophosphate levels, leading to calcification; indeed, levamisole, an inhibitor of TNAP, prevented calcium crystal formation by CD73-deficient fibroblasts. The selective involvement of lower-extremity arteries may be related to the particular distribution of adenosine receptors in these tissues.22
Proposed Mechanism of Mineralization Due to CD73 Deficiency from an NT5E Mutation
Knowledge of the basic defect in our patients allows for consideration of therapeutic interventions. Bisphosphonates, which are pyrophosphate analogues and potent inhibitors of tissue calcification, have been successfully used to treat ENPP1 deficiency and might prove beneficial in patients with CD73 deficiency.23,24
Dipyridamole, an antithrombotic drug used successfully in patients with aneurysmal vascular remodeling, could provide adenosine rescue, since it inhibits cellular reuptake of adenosine (and subsequent degradation by adenosine deaminase) both in vitro and in vivo.25
Other therapeutic possibilities include the use of adenosine-receptor agonists or a direct inhibitor of TNAP such as lansoprazole.26,27
The potential efficacy of such interventions can be investigated in cultured cells, which exhibit both TNAP and calcification phenotypes that are abrogated by transduction with a CD73-encoded lentiviral vector. Mice with CD73 deficiency can also be studied, if a calcification phenotype can be discerned,28,29
to elucidate the role of adenosine in regulating vascular calcification, influencing bone mineralization, and modulating ectopic calcium deposition.
In summary, we identified mutations in NT5E in members of three families with symptomatic arterial and joint calcifications. This gene encodes CD73, a nucleotidase that converts AMP to adenosine. Thus, our results support a role for this metabolic pathway in inhibiting ectopic tissue calcification.