The paradigm for studying such medical mysteries begins with detailed and accurate phenotyping. For example, UDP clinical investigators ascertained a family of five adults from middle America with stunning arterial calcification and claudication confined to the lower extremities (St. Hilaire et al., 2011
). Intense clinical examinations verified that this was not atherosclerosis, that the coronaries were spared, and that the only additional sites of ectopic calcification involved the joint capsules of the hands and feet. Endocrine abnormalities of calcium and phosphate were ruled out.
The next step involved applying genetic methodologies to identify candidate genes causing this disease. In this case, the project benefited enormously from the fact that the parents of the five siblings were third cousins, unaffected, and readily available for study. The most appropriate genetic platform to employ was the million SNP array, which detects copy number variants (deletions and duplications) and also identifies regions of homozygosity. In a product of a consanguineous mating, most homozygous areas derive from a common ancestor, and a single pathogenic mutation in a shared ancestral gene could cause a recessive disease. Hence, we were looking for a region of the human genome in which all five affected siblings were homozygous, but the parents were heterozygous. Only one area satisfied these requirements – a 22-Mb region on chromosome 6. That area contained 92 genes; any one of them could have contained a homozygous mutation that caused the arterial calcifications. We needed an expert in vascular biology to identify the best candidates to pursue.
This was the third, and most crucial, step in discovering a new human disease, i.e. identifying and connecting with a basic scientist whose lab possessed expertise in the field. In this case, Manfred Boehm and Cynthia St. Hilaire of the National Heart, Lung and Blood Institute were authorities on vascular cell metabolism, and cell-based assays were extant in the Boehm lab. In concert with the UDP, whose clinicians provided a skin biopsy, St. Hilaire cultured an affected patient’s fibroblasts and performed expression analysis of candidate genes in the inherited homozygous region. Only one gene, NT5E, which encodes the CD73 protein, was differentially expressed. Direct sequencing of NT5E identified null mutations that resulted in complete inactivation of CD73 protein in all affected siblings. This newly identified vascular disease was referred to as ‘arterial calcification due to deficiency of CD73′ (ACDC). CD73 is a membrane-bound enzyme that converts extracellular AMP to adenosine and inorganic phosphate; this process was found to be severely impaired in fibroblasts from individuals with ACDC. The failure to generate extracellular adenosine leads to an increase in tissue non-specific alkaline phosphatase (TNAP), a key enzyme in tissue calcification in vitro and in vivo. Cultured fibroblasts of individuals with ACDC not only contained increased TNAP activity, but they also calcified. Finally, TNAP activity and in vitro calcification could be reversed by genetic rescue with NT5E cDNA or treatment with exogenous adenosine.
The role of adenosine in inhibiting default calcification in vascular endothelial cells is a new concept that could have an enormous influence on our understanding of ectopic calcification in general. The pathway might also have implications for other specific disorders, including Monckeberg’s medial sclerosis and pseudoxanthoma elasticum (Markello et al., 2011
). In addition, knowledge of the basic defect in ACDC allows for consideration of treatment for affected individuals; besides the five siblings described above, four other ACDC patients have been ascertained worldwide, and still more have come to our attention recently. Treatment with bisphosphonates (inhibitors of alkaline phosphatase) is a potential therapy; therefore, we have initiated a clinical protocol to test this.