X-linked sideroblastic anemia (XLSA) is the most common form of congenital sideroblastic anemia (CSA), and is associated with the mutations in the 5-aminolevulinate synthase 2 (ALAS2). The genetic basis of more than 40% of CSA cases remains unknown.
A two-generation Chinese family with XLSA was studied by next-generation sequencing to identify the underlying CSA-related mutations.
In the study, we identified a missense ALAS2 R204Q mutation in a hemizygous Chinese Han man and in his heterozygous daughter. The male proband presented clinical manifestations at 38 years old and had a good response to pyridoxine.
XLSA, as a hereditary disease, can present clinical manifestations later in lives, for adult male patients with ringed sideroblasts and hypochromic anemia, it should be evaluated with gene analyses to exclude CSA.

X-linked protoporphyria (XLP) (MIM 300752) is an erythropoietic porphyria due to gain-of-function mutations in the last exon (Ducamp et al., Hum Mol Genet 22:1280-88, 2013) of the erythroid-specific aminolevulinate synthase gene (ALAS2). Five ALAS2 exon 11 variants identified by the NHBLI Exome sequencing project (p.R559H, p.E565D, p.R572C, p.S573F and p.Y586F) were expressed, purified and characterized in order to assess their possible contribution to XLP. To further characterize the XLP gain-of-function region, five novel ALAS2 truncation mutations (p.P561X, p.V562X, p.H563X, p.E569X and p.F575X) were also expressed and studied.
Site-directed mutagenesis was used to generate ALAS2 mutant clones and all were prokaryotically expressed, purified to near homogeneity and characterized by protein and enzyme kinetic assays. Standard deviations were calculated for 3 or more assay replicates.
The five ALAS2 single nucleotide variants had from 1.3- to 1.9-fold increases in succinyl-CoA Vmax and 2- to 3-fold increases in thermostability suggesting that most could be gain-of-function modifiers of porphyria instead of causes. One SNP (p.R559H) had markedly low purification yield indicating enzyme instability as the likely cause for XLSA in an elderly patient with x-linked sideroblastic anemia. The five novel ALAS2 truncation mutations had increased Vmax values for both succinyl-CoA and glycine substrates (1.4 to 5.6-fold over wild-type), while the Kms for both substrates were only modestly changed. Of interest, the thermostabilities of the truncated ALAS2 mutants were significantly lower than wild-type, with an inverse relationship to Vmax fold-increase.
Patients with porphyrias should always be assessed for the presence of the ALAS2 gain-of-function modifier variants identified here. A key region of the ALAS2 carboxyterminal region is identified by the truncation mutations studied here and the correlation of increased thermolability with activity suggests that increased molecular flexibility/active site openness is the mechanism of enhanced function of mutations in this region providing further insights into the role of the carboxyl-terminal region of ALAS2 in the regulation of erythroid heme synthesis.

Ring sideroblasts are a hallmark of sideroblastic anemia, although little is known about their characteristics. Here, we first generated mutant mice by disrupting the GATA-1 binding motif at the intron 1 enhancer of the ALAS2 gene, a gene responsible for X-linked sideroblastic anemia (XLSA). Although heterozygous female mice showed an anemic phenotype, ring sideroblasts were not observed in their bone marrow. We next established human induced pluripotent stem cell-derived proerythroblast clones harboring the same ALAS2 gene mutation. Through coculture with sodium ferrous citrate, mutant clones differentiated into mature erythroblasts and became ring sideroblasts with upregulation of metal transporters (MFRN1, ZIP8, and DMT1), suggesting a key role for ferrous iron in erythroid differentiation. Interestingly, holo-transferrin (holo-Tf) did not induce erythroid differentiation as well as ring sideroblast formation, and mutant cells underwent apoptosis. Despite massive iron granule content, ring sideroblasts were less apoptotic than holo-Tf-treated undifferentiated cells. Microarray analysis revealed upregulation of antiapoptotic genes in ring sideroblasts, a profile partly shared with erythroblasts from a patient with XLSA. These results suggest that ring sideroblasts exert a reaction to avoid cell death by activating antiapoptotic programs. Our model may become an important tool to clarify the pathophysiology of sideroblastic anemia.