New Mutation in ALAS2 as the Cause of X-Linked Sideroblastic Anemia Responsive to Pyridoxine: Comparison of ALAS2-Defective and DMT1-Defective BFU-E Growth.

Abstract 4051

Poster Board III-986

Congenital sideroblastic anemias (SA) are hypochromic microcytic anemias with secondary iron overload. Local iron accumulation in mitochondria results in the formation of ringed sideroblasts in the bone marrow. Defects in heme synthesis, Fe-S cluster biogenesis and export have been reported in SA patients. X-linked sideroblastic anemia is a rare gonosomal recessive disorder, resulting from mutations in erythroid-specific delta-aminolevulinate synthase (ALAS2), the first enzyme of heme biosynthesis. It affects mostly males. Few cases of affected females, due to skewed X-inactivation pattern favoring the mutant allele have been reported. Inefficiency of ALAS2 leads to decreased heme production and ineffective erythropoiesis. We investigated a group of 4 young males (average age 28) with congenital SA. Two of the patients had family history of SA and iron overload in males corresponding with the X-linked inheritance. All the patients had hemoglobin level lower than 119 g/L, mean corpuscular volume under 71 fL, increased serum ferritin and numerous sideroblasts in their bone marrow. We performed screening of all exons and promoter region of ALAS2. One patient was negative for ALAS2 mutation, and is a candidate for SLC25A38 mutation screening which only recently has been published as the second most frequent lesion in congenital SA. In two patients we found previously published R452H and R452C mutations; a novel K156E substitution was discovered in one patient. All these three patients responded partially to high dose of pyridoxine. K156 is a conserved amino acid residue and K156E substitution is SIFT predicted as affecting the protein function (the functional study is ongoing). This mutation was not found in 40 healthy controls or 10 patients with myelodysplastic syndrome with SA (RARS, RCMD-RS). Based on our previous study with DMT-1-deficient erythroid progenitors (Priwitzerova et al. Blood 2004;103:3991-2), we also evaluated a possible erythropoietic defect of ALAS2-mutant erythroid progenitors using methylcellulose-based colony assays. We observed that the in vitro growth of all aforementioned ALAS2-mutant patient's erythroid progenitors (BFU-Es) is not affected, which is in contrast to the defective growth of DMT1-mutant erythroid progenitors. These results suggest that impaired heme synthesis is better tolerated by erythroid progenitors than general iron deficiency due to the block in erythroid iron uptake by DMT1 and that the defect in heme synthesis does not fully account for the impaired hemoglobinization and poor growth of DMT1-defective BFU-Es. These data support the role of iron in other processes involved in erythroid-colony development apart from heme synthesis. In conclusion, we present a novel K156E ALAS2 mutation leading to pyridoxine-responsive X-linked sideroblastic anemia. Using BFU-E assays we also submit that lesions in iron-dependent proteins apart from defective heme synthesis contribute to impaired erythroid colony formation in previously described DMT1-mutant erythroid progenitors.

Grant support: Ministry of Health Czech Republic grants NS9935-3 and NS10281-3 and Ministry of Education, Youth and Sports program MSM 6198959205.