Contributions
Abstract: S273
Type: Oral Presentation
Session title: Changing the scene on congenital anemias
Background
5-aminolevulinic acid synthase 2 (ALAS2) is the first and rate-limiting enzyme of erythroid heme biosynthesis. It combines glycine and succinyl-coA to form 5-aminolevulinic acid (ALA), the precursor of porphyrins and heme. ALAS2 is essential in mammals, its expression being precisely regulated during erythropoiesis. Inherited ALAS2 defects cause two rare diseases: X-linked Sideroblastic Anemia (XLSA) and X-linked Protoporphyria (XLPP), due to loss-of-function and gain-of-function mutations, respectively. Male XLSA patients have a microcytic hypochromic anemia of variable severity, characterized by abnormal mitochondrial iron deposits in nucleated and enucleated erythroid cells, termed ring sideroblasts and siderocytes. In two-thirds of patients, the anemia responds to pyridoxine (B6) supplementation. Both male and female XLPP patients develop acute photosensitivity, due to accumulation of free protoporphyrin (PP) in erythrocytes. No curative treatment, other than hematopoietic stem cell transplantation, is available for XLSA or XLPP.
Aims
The lack of ALAS2 mouse models has limited both our understanding of physiopathology, and potential treatment of XLSA and XLPP. We aimed to establish XLSA and XLPP mouse models.
Methods
Employing CRISPR/Cas9 gene targeting technology, we established 3 common XLSA (ALAS2 p.R170H, p.R411H and p.R452H) and 1 XLPP (ALAS2 p.Q548X) mutations in mice. The natural history of the disease was determined for each strain, focusing on blood parameters, free PP levels, and erythropoiesis. We also examined the consequences of vitamin B6 deficient and supplemented diets on each of these parameters.
Results
Four independent injections were performed in E0.5 blastocysts (C57BL/6N) generating a total of founder 45 animals. Animals harboring the expected allele were identified (p.R170H: 3 of 13; p.R411H: 1 of 2; p.R452H: 1 of 14 and Q548X: 4 of 16 animals) and transmitted through the germline. Mutant and control littermate animals have been phenotyped up to one-year of age.
XLSA: Variable numbers of siderocytes are present in peripheral blood smears of animals of all three XLSA lineages as early as 1 week of age and each develops a progressive microcytic, hypochromic anemia.As expected male phenotypes were more severe than female phenotypes, but siderocytes and erythroid defects are also observed in female XLSA carriers. The p.R411H strain has the most severe phenotype. B6 depletion in the p.R411H strain results in a severe ineffective erythropoiesis manifest as extreme splenomegaly and a block in erythroid differentiation.
XLPP: Both XLPP p.Q548X males and females accumulate free PP in erythrocytes. Compared to the Fechm1PAS model (C57BL/6J) of erythroid protoporphyria due to a severe recessive loss-of-function mutation in ferrochelatase, the peak of free PP accumulation occurs earlier in age in XLPP mice. In early stages of the disease (8 weeks), XLPP mice do not develop liver fibrosis.
Conclusion
For the first time, knock-in ALAS2 mouse strains with viable loss and gain of function mutations have been obtained and each expresses the key features of the corresponding human diseases.
Keyword(s): Anemia, Heme, Iron metabolism, Red blood cell
Abstract: S273
Type: Oral Presentation
Session title: Changing the scene on congenital anemias
Background
5-aminolevulinic acid synthase 2 (ALAS2) is the first and rate-limiting enzyme of erythroid heme biosynthesis. It combines glycine and succinyl-coA to form 5-aminolevulinic acid (ALA), the precursor of porphyrins and heme. ALAS2 is essential in mammals, its expression being precisely regulated during erythropoiesis. Inherited ALAS2 defects cause two rare diseases: X-linked Sideroblastic Anemia (XLSA) and X-linked Protoporphyria (XLPP), due to loss-of-function and gain-of-function mutations, respectively. Male XLSA patients have a microcytic hypochromic anemia of variable severity, characterized by abnormal mitochondrial iron deposits in nucleated and enucleated erythroid cells, termed ring sideroblasts and siderocytes. In two-thirds of patients, the anemia responds to pyridoxine (B6) supplementation. Both male and female XLPP patients develop acute photosensitivity, due to accumulation of free protoporphyrin (PP) in erythrocytes. No curative treatment, other than hematopoietic stem cell transplantation, is available for XLSA or XLPP.
Aims
The lack of ALAS2 mouse models has limited both our understanding of physiopathology, and potential treatment of XLSA and XLPP. We aimed to establish XLSA and XLPP mouse models.
Methods
Employing CRISPR/Cas9 gene targeting technology, we established 3 common XLSA (ALAS2 p.R170H, p.R411H and p.R452H) and 1 XLPP (ALAS2 p.Q548X) mutations in mice. The natural history of the disease was determined for each strain, focusing on blood parameters, free PP levels, and erythropoiesis. We also examined the consequences of vitamin B6 deficient and supplemented diets on each of these parameters.
Results
Four independent injections were performed in E0.5 blastocysts (C57BL/6N) generating a total of founder 45 animals. Animals harboring the expected allele were identified (p.R170H: 3 of 13; p.R411H: 1 of 2; p.R452H: 1 of 14 and Q548X: 4 of 16 animals) and transmitted through the germline. Mutant and control littermate animals have been phenotyped up to one-year of age.
XLSA: Variable numbers of siderocytes are present in peripheral blood smears of animals of all three XLSA lineages as early as 1 week of age and each develops a progressive microcytic, hypochromic anemia.As expected male phenotypes were more severe than female phenotypes, but siderocytes and erythroid defects are also observed in female XLSA carriers. The p.R411H strain has the most severe phenotype. B6 depletion in the p.R411H strain results in a severe ineffective erythropoiesis manifest as extreme splenomegaly and a block in erythroid differentiation.
XLPP: Both XLPP p.Q548X males and females accumulate free PP in erythrocytes. Compared to the Fechm1PAS model (C57BL/6J) of erythroid protoporphyria due to a severe recessive loss-of-function mutation in ferrochelatase, the peak of free PP accumulation occurs earlier in age in XLPP mice. In early stages of the disease (8 weeks), XLPP mice do not develop liver fibrosis.
Conclusion
For the first time, knock-in ALAS2 mouse strains with viable loss and gain of function mutations have been obtained and each expresses the key features of the corresponding human diseases.
Keyword(s): Anemia, Heme, Iron metabolism, Red blood cell