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No Enzyme Activity of 25-Hydroxyvitamin D₃ 1-Hydroxylase Gene Product in Pseudovitamin D Deficiency Rickets, Including That with Mild Clinical Manifestation

Institute of Molecular and Cellular Biosciences (S.Ki., A.M., K.T., S.Ka.), and the Department of Laboratory Medicine (S.F.), The University of Tokyo, 113-0032 Tokyo; the Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University (T.S., K.I.), 606-8502 Kyoto; the Department of Pediatrics, Okayama University Medical School (Y.S.), 700-0914 Okayama; the Department of Pediatrics, Osaka University Medical School (M.S.), 565-0871 Osaka; the Department of Pediatrics and Child Health, Kurume University Medical School (S.Y.), 830-0011 Fukuoka; the Division of Metabolism, Chiba Children’s Hospital (M.T.), 266-0007 Chiba; the Department of Pediatrics, Chiba University School of Medicine (H.N.), 260-8677 Chiba; and Core Research for Evolutional Science and Technology, Japan Science and Technology Corp. (S.Ka.), 332-0012 Saitama, Japan

Address all correspondence and requests for reprints to: Shigeaki Kato, Ph.D., Institute of Molecular and Cellular Biosciences, University of Tokyo, 1–1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan. E-mail: uskato{at}hongo.ecc.u-tokyo.ac.jp

Pseudovitamin D deficiency rickets (PDDR) is an autosomal recessive disorder caused by defect in the activation of vitamin D. We recently isolated 25-hydroxyvitamin D₃ 1-hydroxylase gene and identified four homozygous inactivating missense mutations in this gene by analysis of four typical cases of PDDR. This disease shows some phenotypic variation, and it has been suspected that patients with mild phenotypes have mutations that do not totally abolish the enzyme activity. To investigate the molecular defects associated with the phenotypic variation, we analyzed six additional unrelated PDDR patients: one with mild and five with typical clinical manifestation. By sequence analysis, all six patients were proven to have mutations in both alleles. The mutations varied, and we identified four novel missense mutations, a nonsense mutation, and a splicing mutation for the first time. The patient with mild clinical symptoms was compound heterozygous for T321R and a splicing mutation. The splice site mutation caused intron retention. Enzyme activity of the T321R mutant was analyzed by overexpressing the mutant 1-hydroxylase in Escherichia coli cells to detect the subtle residual enzyme activity. No residual enzyme activity was detected in T321R mutant or in the other mutants. These results indicate that all of the patients, including those of mild phenotype, are caused by 1-hydroxylase gene mutations that totally abolish the enzyme activity.

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