NT5E mutations and arterial calcifications

Abstract

Background: Arterial calcifications are associated with increased cardiovascular risk, but the genetic basis of this association is unclear.

Methods: We performed clinical, radiographic, and genetic studies in three families with symptomatic arterial calcifications. Single-nucleotide-polymorphism analysis, targeted gene sequencing, quantitative polymerase-chain-reaction assays, Western blotting, enzyme measurements, transduction rescue experiments, and in vitro calcification assays were performed.

Results: We identified nine persons with calcifications of the lower-extremity arteries and hand and foot joint capsules: all five siblings in one family, three siblings in another, and one patient in a third family. Serum calcium, phosphate, and vitamin D levels were normal. Affected members of Family 1 shared a single 22.4-Mb region of homozygosity on chromosome 6 and had a homozygous nonsense mutation (c.662C→A, p.S221X) in NT5E, encoding CD73, which converts AMP to adenosine. Affected members of Family 2 had a homozygous missense mutation (c.1073G→A, p.C358Y) in NT5E. The proband of Family 3 was a compound heterozygote for c.662C→A and c.1609dupA (p.V537fsX7). All mutations found in the three families result in nonfunctional CD73. Cultured fibroblasts from affected members of Family 1 showed markedly reduced expression of NT5E messenger RNA, CD73 protein, and enzyme activity, as well as increased alkaline phosphatase levels and accumulated calcium phosphate crystals. Genetic rescue experiments normalized the CD73 and alkaline phosphatase activity in patients' cells, and adenosine treatment reduced the levels of alkaline phosphatase and calcification.

Conclusions: We identified mutations in NT5E in members of three families with symptomatic arterial and joint calcifications. This gene encodes CD73, which converts AMP to adenosine, supporting a role for this metabolic pathway in inhibiting ectopic tissue calcification. (Funded by the National Human Genome Research Institute and the National Heart, Lung, and Blood Institute of the National Institutes of Health.).

Figure 1. Pedigrees of the Study Patients and Radiographic Findings

Panel A shows the pedigrees of the three study families. Open symbols indicate unaffected family members, and solid red symbols affected members. Arrows indicate the probands. Squares indicate male family members, circles female members, slashes deceased members, and double horizontal lines consanguinity. The diamond indicates offspring of unknown number, and the triangle a lost pregnancy. Panel B shows plain radiographs of popliteal arteries of the three probands. Panel C shows plain radiographs of the pelvis and femurs (left) and ankle (right) of Patient VI.1 of Family 1, revealing calcified arteries (arrow). Panel D shows radiographs of metacarpal phalangeal and interphalangeal joint calcification (arrow) in Patient VI.1 of Family 1.

Figure 2. Results of Genetic and Enzyme Studies in Family 1

Panel A shows single-nucleotide polymorphism (SNP)–array homozygosity plots for the five affected siblings in Family 1 and their parents. The five affected siblings shared a region in which heterozygosity was absent, reflecting identity by means of descent in this consanguineous family. Panel B shows sequence chromatograms for a control, a parent of an affected member of Family 1, and an affected member; the nonsense mutation (p.S221X) is indicated. Panel C shows NT5E messenger RNA (complementary DNA [cDNA]) expression in Patients VI.4 and VI.1 as compared with controls. Panel D shows a deficiency in CD73 enzyme activity (represented by AMP-dependent inorganic phosphate production) in cultured fibroblasts from Patient VI.4 and Patient VI.1 of Family 1. Panel E shows CD73 enzyme activity in fibroblasts from controls and from Patient VI.4 after the fibroblasts were transduced with either an empty vector (containing green fluorescent protein only) or a CD73-containing vector. The CD73 vector increased CD73 activity (represented by AMP-dependent inorganic phosphate production) in both the control cells and the patients’ cells. Panel F shows CD73 activity (represented by AMP-dependent inorganic phosphate production) in HEK293 cells transfected with an empty vector or a vector containing wild-type NT5E or NT5E with the c.622C→A, c.1073G→A, or c.1609dupA mutation or both the c.622C→A and c.1609dupA mutations (in a 1:1 ratio, simulating the state in Patient II.1 of Family 3). In Panels C, D, and F, data are the means of three experiments and were analyzed with the use of one-way analysis of variance with Dunnett’s multiple-comparison post hoc test. In Panel E, data are the means of three experiments and were analyzed with the use of an unpaired Student’s t-test. P values are shown for the comparison with controls in Panels C and D and with the various mutated cDNA in Panel F. In Panels C through F, I bars indicate the standard deviations.

Figure 3. Studies of Fibroblasts Obtained from Patient VI.4 of Family 1

Panel A shows the results of staining for tissue-nonspecific alkaline phosphatase (TNAP) in fibroblasts from a control and from Patient VI.4 after incubation for 3 days in calcifying medium. The increased staining in the patient’s cells (middle image) was reduced by adding 30 µM adenosine to the incubation medium daily (right image). Panel B shows TNAP activity in fibroblasts from a control and from Patient VI.4 after incubation for 3 days in calcifying medium. Transduction with a control vector expressing β-galactosidase had little effect on alkaline phosphatase activity, whereas transduction with a CD73-encoding vector reduced alkaline phosphatase activity significantly; incubation in 30 µM adenosine produced TNAP levels similar to those seen in control cells. The data shown are the means of three experiments and were analyzed by means of analysis of variance with Dunnett’s multiple-comparison post hoc test. P values are shown for the comparison with no treatment. I bars indicate standard deviations. Panel C shows the effects of interventions on calcium phosphate crystal formation in fibroblasts from a control and from Patient VI.4. Staining for calcium with alizarin red S showed abundant staining in the patient’s cells, as compared with no staining in control cells, after 21 days in calcifying medium. Calcium staining was prevented by transduction with a CD73-encoding lentiviral vector, but not a control vector expressing β-galactosidase, and by treatment every fourth day with 1 mM levamisole; daily treatment with 30 µM adenosine partially abrogated the calcification process.

Figure 4. Proposed Mechanism of Mineralization Due to CD73 Deficiency from an NT5E Mutation

On the surface of vascular cells, ENPP1 (the protein encoded by the ectonucleotide pyrophosphatase–phosphodiesterase 1 gene) converts ATP to AMP and pyrophosphate (PPi), and CD73 converts AMP to adenosine and inorganic phosphate (Pi). Pyrophosphate inhibits calcification, tissue-nonspecific alkaline phosphatase (TNAP) degrades pyrophosphate, and adenosine inhibits TNAP. Deficiency of CD73 results in decreased adenosine levels, eliminating the inhibition of TNAP from the pathway either directly or by way of adenosine receptor signaling. Increased TNAP from the pathway activity results in decreased pyrophosphate and increased cell calcification.