Fetal compensation of the hemolytic anemia in mice homozygous for the normoblastosis (nb) mutation

The mouse autosomal recessive mutation nb causes a deficiency of erythroid ankyrin and generates a life-threatening hemolytic anemia in adult mice; however, at birth, nb/nb mice appear to be robust and show no pallor. In our study, the time of disease onset was sought by comparison of nb/nb and +/? mice both in utero and postnatally. Erythroid ankyrin messenger RNA (mRNA) is expressed in fetal erythroid progenitors from normal mice, but is reduced to 10% of normal levels in mutant fetuses. Despite the deficiency of erythroid ankyrin mRNA, 16 and 18 day nb/nb fetuses have normal levels of red blood cells (RBCs) and the RBCs are morphologically normal by scanning electron microscopy. The earliest signs of any clinical anomaly are an increase in the number of circulating reticulocytes and the deposition of minor amounts of iron just before birth in the 18 day fetal nb/nb liver, suggesting that RBCs are being destroyed. Within 24 hours after birth, nb/nb neonates have a slight but significant decrease of their RBC counts. During the next 5 days, the nb/nb RBC counts decrease markedly, the reticulocyte counts assume the mutant adult levels of 60%, the erythrocytes become microcytic and fragmented, and iron deposits accumulate in the liver. The rapid onset of clinical disease postnatally, coupled with our findings that the erythroid ankyrin gene is transcribed in fetal erythroid cell precursors from normal mice, suggest that mechanisms exist in the nb/nb fetus to compensate for the erythroid ankyrin deficiency.</jats:p

Fetal compensation of the hemolytic anemia in mice homozygous for the normoblastosis (nb) mutation

Abstract The mouse autosomal recessive mutation nb causes a deficiency of erythroid ankyrin and generates a life-threatening hemolytic anemia in adult mice; however, at birth, nb/nb mice appear to be robust and show no pallor. In our study, the time of disease onset was sought by comparison of nb/nb and +/? mice both in utero and postnatally. Erythroid ankyrin messenger RNA (mRNA) is expressed in fetal erythroid progenitors from normal mice, but is reduced to 10% of normal levels in mutant fetuses. Despite the deficiency of erythroid ankyrin mRNA, 16 and 18 day nb/nb fetuses have normal levels of red blood cells (RBCs) and the RBCs are morphologically normal by scanning electron microscopy. The earliest signs of any clinical anomaly are an increase in the number of circulating reticulocytes and the deposition of minor amounts of iron just before birth in the 18 day fetal nb/nb liver, suggesting that RBCs are being destroyed. Within 24 hours after birth, nb/nb neonates have a slight but significant decrease of their RBC counts. During the next 5 days, the nb/nb RBC counts decrease markedly, the reticulocyte counts assume the mutant adult levels of 60%, the erythrocytes become microcytic and fragmented, and iron deposits accumulate in the liver. The rapid onset of clinical disease postnatally, coupled with our findings that the erythroid ankyrin gene is transcribed in fetal erythroid cell precursors from normal mice, suggest that mechanisms exist in the nb/nb fetus to compensate for the erythroid ankyrin deficiency.</jats:p

Complete nucleotide sequence of the murine erythroid beta-spectrin cDNA and tissue-specific expression in normal and jaundiced mice

Spectrin, a heterodimer of alpha and beta subunits, is an essential component of the red blood cell membrane skeleton. The jaundiced (ja/ja) mutation causes a severe hemolytic anemia in mice and is mapped to the erythroid beta-spectrin locus (Spnb-1) on chromosome 12. As a prerequisite for determining the molecular defect of the jaundiced mutation, we have cloned and sequenced the complete murine reticulocyte cDNA for normal Spnb-1. Two unique transcripts that differ in the placement of polyA tails are represented in the clones isolated. Amino acid sequence comparison between erythroid and murine brain spectrin (Spnb-2, chromosome 11) shows 67% identity throughout repeats 16 and 17 and complete divergence in domain III, which is associated with the alpha/beta subunit dimerization and phosphorylation. We examined the tissue distribution of normal and mutant erythroid beta-spectrin transcripts using domain-specific probes. Transcripts are detected in normal spleen tissue and reticulocytes (8 and 9.6 kb), brain tissue (10 and 11 kb), skeletal muscle tissue, and cardiac muscle tissue (11, 10.3, 7.2, and 4.0 kb). Extensive variability in mRNA processing is shown with region-specific probes. Steady state levels of the mutant transcripts are reduced when hybridized with a probe to repeats 2 through 6 with the exception of the 7.2-kb transcript that is unique to heart and skeletal muscle tissues, and is present at normal and elevated levels, respectively, in ja/ja mice. These results provide evidence for more diverse tissue-specific products of the Spnb-1 gene than were previously suspected.</jats:p

Complete nucleotide sequence of the murine erythroid beta-spectrin cDNA and tissue-specific expression in normal and jaundiced mice

Abstract Spectrin, a heterodimer of alpha and beta subunits, is an essential component of the red blood cell membrane skeleton. The jaundiced (ja/ja) mutation causes a severe hemolytic anemia in mice and is mapped to the erythroid beta-spectrin locus (Spnb-1) on chromosome 12. As a prerequisite for determining the molecular defect of the jaundiced mutation, we have cloned and sequenced the complete murine reticulocyte cDNA for normal Spnb-1. Two unique transcripts that differ in the placement of polyA tails are represented in the clones isolated. Amino acid sequence comparison between erythroid and murine brain spectrin (Spnb-2, chromosome 11) shows 67% identity throughout repeats 16 and 17 and complete divergence in domain III, which is associated with the alpha/beta subunit dimerization and phosphorylation. We examined the tissue distribution of normal and mutant erythroid beta-spectrin transcripts using domain-specific probes. Transcripts are detected in normal spleen tissue and reticulocytes (8 and 9.6 kb), brain tissue (10 and 11 kb), skeletal muscle tissue, and cardiac muscle tissue (11, 10.3, 7.2, and 4.0 kb). Extensive variability in mRNA processing is shown with region-specific probes. Steady state levels of the mutant transcripts are reduced when hybridized with a probe to repeats 2 through 6 with the exception of the 7.2-kb transcript that is unique to heart and skeletal muscle tissues, and is present at normal and elevated levels, respectively, in ja/ja mice. These results provide evidence for more diverse tissue-specific products of the Spnb-1 gene than were previously suspected.</jats:p

Distinct fetal Ank-1 and Ank-2 related proteins and mRNAs in normal and nb/nb mice

Mice homozygous for the mutation normoblastosis (gene symbol nb on chromosome 8) are deficient in erythroid ankyrin (ANK-1) and have a severe hemolytic anemia throughout life. Characteristic of the disease is a dramatic decrease in the level of expression of the Ank-1 gene (chromosome 8). The other major ankyrin transcript, brain ankyrin (Ank- 2 on chromosome 3) is expressed at normal levels in nb/nb mice. Surprisingly, nb/nb fetuses have normal erythrocyte counts despite the decreased levels of Ank-1 transcripts. We previously hypothesized that fetal-specific ankyrin-related proteins could exist in nb/nb fetuses to account for the lack of detrimental effects of ANK-1 deficiency. In the present report, Western and Northern blot analyses were performed on hematopoietic cells isolated from nb/nb and +/+ fetuses. An ANK-1- related protein (165 Kd) in fetal reticulocytes persisted in adult nb/nb but not in +/+ reticulocytes. An Ank-1-related transcript of 5.5 kb was found in fetal reticulocytes. This transcript appeared to be upregulated in nb/nb but not in +/+ adult reticulocytes. A fetal- specific ANK-2-related protein (155 Kd) was present in nb/nb and in +/+ fetal reticulocytes. Ank-2-related fetal liver mRNAs were present during the time the liver was actively generating erythrocytes. Neither the Ank-2-related transcripts nor the 155-Kd ANK-2-related protein were found in +/+ or mutant adult reticulocytes. The data indicate that (1) unique ankyrin-related proteins and mRNAs present in fetal erythrocytes may stabilize the ankyrin-deficient nb/nb erythrocytes and (2) adult nb/nb mice may upregulate fetal gene transcripts to compensate for the ANK-1 deficiency.</jats:p

Distinct fetal Ank-1 and Ank-2 related proteins and mRNAs in normal and nb/nb mice

Abstract Mice homozygous for the mutation normoblastosis (gene symbol nb on chromosome 8) are deficient in erythroid ankyrin (ANK-1) and have a severe hemolytic anemia throughout life. Characteristic of the disease is a dramatic decrease in the level of expression of the Ank-1 gene (chromosome 8). The other major ankyrin transcript, brain ankyrin (Ank- 2 on chromosome 3) is expressed at normal levels in nb/nb mice. Surprisingly, nb/nb fetuses have normal erythrocyte counts despite the decreased levels of Ank-1 transcripts. We previously hypothesized that fetal-specific ankyrin-related proteins could exist in nb/nb fetuses to account for the lack of detrimental effects of ANK-1 deficiency. In the present report, Western and Northern blot analyses were performed on hematopoietic cells isolated from nb/nb and +/+ fetuses. An ANK-1- related protein (165 Kd) in fetal reticulocytes persisted in adult nb/nb but not in +/+ reticulocytes. An Ank-1-related transcript of 5.5 kb was found in fetal reticulocytes. This transcript appeared to be upregulated in nb/nb but not in +/+ adult reticulocytes. A fetal- specific ANK-2-related protein (155 Kd) was present in nb/nb and in +/+ fetal reticulocytes. Ank-2-related fetal liver mRNAs were present during the time the liver was actively generating erythrocytes. Neither the Ank-2-related transcripts nor the 155-Kd ANK-2-related protein were found in +/+ or mutant adult reticulocytes. The data indicate that (1) unique ankyrin-related proteins and mRNAs present in fetal erythrocytes may stabilize the ankyrin-deficient nb/nb erythrocytes and (2) adult nb/nb mice may upregulate fetal gene transcripts to compensate for the ANK-1 deficiency.</jats:p