NT5E Mutations and Arterial Calcifications
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T h e new england journal o f medicine original article NT5E Mutations and Arterial Calcifications Cynthia St. Hilaire, Ph.D., Shira G. Ziegler, B.A., Thomas C. Markello, M.D., Ph.D., Alfredo Brusco, Ph.D., Catherine Groden, M.S., Fred Gill, M.D., Hannah Carlson-Donohoe, B.A., Robert J. Lederman, M.D., Marcus Y. Chen, M.D., Dan Yang, M.D., Ph.D., Michael P. Siegenthaler, M.D., Carlo Arduino, M.D., Cecilia Mancini, M.Sc., Bernard Freudenthal, M.D., Horia C. Stanescu, M.D., Anselm A. Zdebik, M.D., Ph.D., R. Krishna Chaganti, M.D., Robert L. Nussbaum, M.D., Robert Kleta, M.D., Ph.D., William A. Gahl, M.D., Ph.D., and Manfred Boehm, M.D. ABSTRACT BACKGROUND From the National Heart, Lung, and Arterial calcifications are associated with increased cardiovascular risk, but the ge- Blood Institute (C.S.H., R.J.L., M.Y.C., netic basis of this association is unclear. D.Y., M.P.S., M.B.), the National Human Genome Research Institute (S.G.Z., T.C.M., C.G., H.C.-D., W.A.G.), the National Insti- METHODS tutes of Health (NIH) Undiagnosed Dis- We performed clinical, radiographic, and genetic studies in three families with symp- eases Program and the Office of Rare Diseases Research (T.C.M., C.G., W.A.G.), tomatic arterial calcifications. Single-nucleotide-polymorphism analysis, targeted and the Clinical Center (F.G.) — all at the gene sequencing, quantitative polymerase-chain-reaction assays, Western blotting, NIH, Bethesda, MD; Azienda Ospedali- enzyme measurements, transduction rescue experiments, and in vitro calcification era Universitaria San Giovanni Battista, Struttura Complessa a Direzione Univer- assays were performed. sitaria Medical Genetics, and the Depart- ment of Genetics, Biology, and Biochem- RESULTS istry, University of Turin, Turin, Italy (A.B., C.A., C.M.); the Departments of We identified nine persons with calcifications of the lower-extremity arteries and Medicine and Physiology, University Col- hand and foot joint capsules: all five siblings in one family, three siblings in an- lege London, London (B.F., H.C.S., other, and one patient in a third family. Serum calcium, phosphate, and vitamin D A.A.Z., R.K.); and the Department of Medicine (R.K.C.) and the Institute of levels were normal. Affected members of Family 1 shared a single 22.4-Mb region Human Genetics (R.L.N.), University of of homozygosity on chromosome 6 and had a homozygous nonsense mutation California, San Francisco, San Francisco. (c.662C→A, p.S221X) in NT5E, encoding CD73, which converts AMP to adenosine. Address reprint requests to Dr. Gahl at the NIH Undiagnosed Diseases Pro- Affected members of Family 2 had a homozygous missense mutation (c.1073G→A, gram, 10 Center Dr., Bldg. 10, Rm. 10C- p.C358Y) in NT5E. The proband of Family 3 was a compound heterozygote for 103, National Human Genome Research c.662C→A and c.1609dupA (p.V537fsX7). All mutations found in the three families Institute, National Institutes of Health, Bethesda, MD 20892, or at bgahl@helix result in nonfunctional CD73. Cultured fibroblasts from affected members of Fam- .nih.gov. ily 1 showed markedly reduced expression of NT5E messenger RNA, CD73 protein, and enzyme activity, as well as increased alkaline phosphatase levels and accumu- Dr. St. Hilaire, Ms. Ziegler, and Drs. Markello, Kleta, Gahl, and Boehm con- lated calcium phosphate crystals. Genetic rescue experiments normalized the CD73 tributed equally to this article. and alkaline phosphatase activity in patients’ cells, and adenosine treatment reduced the levels of alkaline phosphatase and calcification. N Engl J Med 2011;364:432-42. Copyright © 2011 Massachusetts Medical Society. 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.) 432 n engl j med 364;5 nejm.org february 3, 2011 NT5E mutations and arterial calcifications ascular calcification, arising ei- Analysis of single-nucleotide polymorphisms ther in the intima or media of vessels, is (SNPs) associated with an excess risk of cardio- Genomic DNA in Family 1 was isolated from pe- V 1,2 vascular events. This was initially considered a ripheral leukocytes and genotyped on a genotyp- passive response to degenerative events, but mount- ing array (Human 1M Duo, Illumina). Homozy- ing evidence suggests it is the result of a process gosity-allele plots were generated with the use of that mimics active bone remodeling.3,4 Extracel- GenomeStudio software. Anomalous regions of lular calcification is increasingly viewed as arising homozygosity were identified visually and con- from a default biochemical pathway and requir- firmed by means of haplotype imputation (ENT ing the constant stimulation of inhibitory systems program). A lod score was established with the to prevent its occurrence. use of parametric multipoint linkage analysis, as Only a single mendelian disorder of isolated previously described.7 vascular calcification — idiopathic infantile arte- rial calcification, now also referred to as gener- Mutation Analysis of CD73 alized arterial calcification of infancy — has been Coding exons and intron–exon junctions of NT5E described.5 This autosomal recessive disease, due were amplified with the use of a touchdown poly- to mutations in the ectonucleotide pyrophospha- merase-chain-reaction (PCR) assay of 50 ng of tase–phosphodiesterase 1 gene (ENPP1), often re- genomic DNA, 3 μM of sense and antisense oli- sults in death during childhood, apparently ow- gonucleotides, and 5 μl of HotStart Master Mix ing to disruption of the trophic influences that (Qiagen) in a final volume of 10 μl. PCR products inhibit vascular-cell calcification. We conducted a were sequenced in both directions with the use study to evaluate an adult-onset disorder in three of the Big Dye terminator kit (version 1.1, Applied families whose affected members had extensive Biosystems) and an automated capillary sequenc- calcifications of the lower-extremity arteries and er (ABI PRISM 3130xl Genetic Analyzer, Applied small joint capsules and to investigate a possible Biosystems). We compared electrophoretogram- genetic basis of the symptoms. derived sequences with reference sequences for NT5E (Ensembl gene number ENSG00000135318) Methods by using Sequencher software (version 4.8). Screen- ing of 200 DNA samples from controls of white Patients race (panel HD200CAU, Coriell Cell Repositories) Three families were studied. Members of Family was performed by means of the 5′−nuclease al- 1 and Family 3 were admitted to the National In- lelic discrimination (TaqMan) assay, as previ- stitutes of Health (NIH) Undiagnosed Diseases ously described.8 Details of the PCR amplifica- Program and enrolled in clinical studies whose tion, primer sequences, and allelic discrimination protocols had been approved by the institutional assay are available in the Supplementary Appen- review board of the National Human Genome Re- dix, available with the full text of this article at search Institute. The genetic studies for Family 2 NEJM.org. were approved by the institutional review board of Azienda Ospedaliera Universitaria San Giovanni Expression Studies Battista. All subjects provided written informed RNA was isolated from cultured fibroblasts with consent. the use of the RNeasy kit (Qiagen), and comple- mentary DNA (cDNA) was generated from 1-μg Fibroblast-Cell Cultures RNA samples with the use of the SuperScript II Fibroblast cultures were prepared from a 4-mm Reverse Transcriptase kit (Invitrogen). Expression punch-biopsy skin specimen obtained from each of NT5E was measured by means of a quantitative patient and grown in Dulbecco’s modified Eagle’s real-time PCR assay involving SYBR Green tech- medium containing 10% fetal-calf serum and 1% nology on a Chromo4 Real Time PCR Detection penicillin–streptomycin, as previously described.6 System (Bio-Rad). Expression levels were calculat- Cells were fed every other day and split 1:2 at con- ed on the basis of the 2−ΔCt method, in which the fluence. cycling threshold (Ct) of the candidate gene is n engl j med 364;5 nejm.org february 3, 2011 433 T h e new england journal o f medicine compared with the Ct of 18S ribosomal RNA and pCMV-Sport6 containing green fluorescent pro- expressed as a power of two (2(Ct of CD73−Ct of 18S)). tein, wild-type NT5E cDNA, or mutated NT5E cDNA Primer sequences for NT5E and 18S rRNA are was transfected into HEK293 cells with the use provided in the Supplementary Appendix. of FuGENE 6 reagent (Roche) according to the manufacturer’s instructions. Three days after Western Blotting for CD73 transfection, cells were analyzed for CD73 activ- Western blotting methods are described in the ity, as described above. Supplementary Appendix. In Vitro Alkaline Phosphatase and Calcium CD73 Enzyme Assay Assays Fibroblasts were washed with a solution of 2 mM Staining for alkaline phosphatase was performed magnesium chloride, 120 mM sodium chloride, by means of the Alkaline Phosphatase Detection 5 mM potassium chloride, 10 mM glucose, and Kit (Millipore). Assay of alkaline phosphatase was 20 mM HEPES. Incubation buffer, consisting of performed with the use of the Quantitative Alka- the wash solution supplemented with 2 mM AMP, line Phosphatase ES Characterization Kit (Milli- was added, and cells were incubated at 37°C for pore) according to the manufacturer’s instruc- 10 minutes.9 The supernatant was removed, and tions. Briefly, cells were trypsinized and collected inorganic phosphate was measured with the in aliquots of 60,000 cells per reaction in p-nitro- SensoLyte MG Phosphate Assay Kit (AnaSpec) ac- phenol–phosphate substrate. Alkaline phospha- cording to the manufacturer’s instructions.