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Vitamin D deficiency remains a major cause of rickets worldwide. Nutritional factors are the major cause and less commonly, inheritance causes. Recently, CYP2R1 has been reported as a major factor for 25-hydroxylation contributing to the inherited forms of vitamin D deficiency. We conducted a prospective cohort study at King Faisal Specialist Hospital Research Centre, Riyadh, Saudi Arabia, to review cases with 25-hydroxylase deficiency and describe their clinical, biochemical, and molecular genetic features. We analyzed 27 patients