X-linked macrocytic dyserythropoietic anemia in females with an ALAS2 mutation

Abstract

Macrocytic anemia with abnormal erythropoiesis is a common feature of megaloblastic anemias, congenital dyserythropoietic anemias, and myelodysplastic syndromes. Here, we characterized a family with multiple female individuals who have macrocytic anemia. The proband was noted to have dyserythropoiesis and iron overload. After an extensive diagnostic evaluation that did not provide insight into the cause of the disease, whole-exome sequencing of multiple family members revealed the presence of a mutation in the X chromosomal gene ALAS2, which encodes 5'-aminolevulinate synthase 2, in the affected females. We determined that this mutation (Y365C) impairs binding of the essential cofactor pyridoxal 5'-phosphate to ALAS2, resulting in destabilization of the enzyme and consequent loss of function. X inactivation was not highly skewed in wbc from the affected individuals. In contrast, and consistent with the severity of the ALAS2 mutation, there was a complete skewing toward expression of the WT allele in mRNA from reticulocytes that could be recapitulated in primary erythroid cultures. Together, the results of the X inactivation and mRNA studies illustrate how this X-linked dominant mutation in ALAS2 can perturb normal erythropoiesis through cell-nonautonomous effects. Moreover, our findings highlight the value of whole-exome sequencing in diagnostically challenging cases for the identification of disease etiology and extension of the known phenotypic spectrum of disease.

Figure 3. X-linked dominant LOF ALAS2 mutations can result in macrocytosis and dyserythropoiesis through cell-nonautonomous effects.

(A) Sequencing of cDNA derived from cultured erythroid cells from the proband showed skewing toward the WT ALAS2 allele at the late stages of erythroid differentiation. Cells began as progenitors (day 5), became intermediate erythroblasts (day 9), and transitioned to orthochromatic erythroblasts (day 14). By late erythropoiesis, only a trace amount of the mutant allele was detectable. (B) Model showing how developing erythroid progenitors and precursors compete for space within the BM. The active X chromosome expressed in each group of cells (brown and pink for the mutant and WT, respectively) is shown in blue, with some cells expressing the mutant (red) and others expressing the WT (green) allele.

Figure 2. Severe LOF with the ALAS2 Y365C mutation and lack of highly skewed X inactivation in female mutation carriers.

(A) Model of ALAS2 shows PLP highlighted in blue and the Y or C amino acid at position 365 highlighted in red. (B) SDS-PAGE gel of WT and mutant ALAS2. Lanes 1 and 8 contain the protein standards, while lanes 2–4 and 5–7 contain WT and mutant ALAS2 protein samples, respectively. Lanes 2 and 5 show partially purified samples after amylose affinity chromatography, lanes 3 and 6 show results after factor Xa digestion, and lanes 4 and 7 show results after gel filtration chromatography. Thin vertical lines in this composite figure separate noncontiguous lanes in the 2 original gels. (C) Chromatographic profiles for purification of WT and mutant ALAS2 by size exclusion (absorbance at 280 nm is shown in milliabsorbance units [mAU]). (D) Quantification of HUMARA results in all affected individuals showing WT and mutant X chromosomes. (E) Sanger sequencing traces of genomic DNA (gDNA) and cDNA derived from reticulocyte mRNA from the proband (II-2) for ALAS2, with an arrow highlighting the mutation.

Figure 1. Identification of an ALAS2 mutation in a family with macrocytic anemia and dyserythropoiesis.

(A) BM aspirate from the proband (II-2) with erythroid hyperplasia. (B) Prussian blue staining revealed rare erythroblasts with siderotic granules. (C) BM aspirate with dyserythropoiesis. (D) Core biopsy demonstrating erythroid hyperplasia and dyserythropoiesis. (E) Pedigree of the family, with affected individuals highlighted by half-filled circles. Arrows highlight those individuals who underwent whole-exome sequencing. (F) Sanger sequencing traces around X chromosome position 55042086 (hg19 coordinates) in 2 affected individuals. Original magnification, ×100 (A and B) and ×2000 (C and D).