Identification of XLSA in a family harboring the heterozygous ALAS2-R227C mutation. (A) Pedigree of the family. Squares indicate males, and circles indicate females. Filled circles denote the patients confirmed by ALAS2 sequencing. The arrow indicates the proband. (B) Sanger sequencing data of ALAS2 genomic DNA from SA2 buccal cells (top) and ALAS2 cDNA from SA2 peripheral blood erythrocytes (bottom) are shown. (C) Morphology of SA2 and SA3 bone marrow cells. An increased number of ring sideroblasts was observed with Prussian blue staining (left). The arrows indicate ring sideroblasts. Megaloblastic change was detected by May Grunwald-Giemsa staining (middle and right). Magnification of the objective lens: ×100 (left and middle) and ×40 (right). Bars represent 25 μm. (D) Summary of HUMARA assays of CD34⁺ and CD235a⁺ bone marrow cells and ALAS2 cDNA Sanger sequencing of peripheral blood erythrocytes. The schematic diagram of erythropoiesis was created using BioRender.com. cDNA, complementary DNA.