Hemolytic anemia in the mouse. Report of a new mutation and clarification of its genetics

A new mutation causing spherocytic, hemolytic anemia has been discovered in the house mouse. It is inherited as a single autosomal recessive gene, allelic with both sph and ha, which, in turn, were shown to be allelic with each other. A revised nomenclature for the three apparent alleles is proposed: sph (formerly sph), sphha (formerly ha), and sph2Bc (the new mutation). Like the other murine hemolytic anemias, sph2Bc involves a defect in the red blood cell membrane protein, spectrin.     

Juvenile spermatogonial depletion (jsd): a genetic defect of germ cell proliferation of male mice

Adult C57BL/6J male mice homozygous for the mutant gene, juvenile spermatogonial depletion (jsd/jsd), show azoosper4ia and testes reduced to one-third normal size, but are otherwise phenotypically normal. In contrast, adult jsd/jsd females are fully fertile. This feature facilitated mapping the jsd gene to the centromeric end of chromosome 1; the gene order is jsd-Isocitrate dehydrogenase-1 (Idh-1)-Peptidase-3 (Pep-3). Analysis of testicular histology from jsd/jsd mice aged 3-10 wk revealed that these mutant mice experience one wave of spermatogenesis, but fail to continue mitotic proliferation of type A spermatogonial cells at the basement membrane. As a consequence, histological sections of testes from mutant mice aged 8-52 wk showed tubules populated by modest numbers of Sertoli cells, with only an occasional spermatogonial cell. Some sperm with normal morphology and motility were observed in epididymides of 6.5- but not in 8-wk or older mutants. Treatment with retinol failed to alter the loss of spermatogenesis in jsd/jsd mice. Analyses of serum hormones of jsd/jsd males showed that testosterone levels were normal at all ages--a finding corroborated by normal seminal vesicle and vas deferens weights, whereas serum follicle-stimulating hormone levels were significantly elevated in mutant mice from 4 to 20 wk of age. We hypothesize the jsd/jsd male may be deficient in proliferative signals from Sertoli cells that are needed for spermatogenesis.     

A Mutation Affecting the Lactate Dehydrogenase Locus Ldh-1 in the Mouse. II. Mechanism of the Ldh-a Deficiency Associated with Hemolytic Anemia

A procarbazine hydrochloride-induced mutation at the Ldh-1 structural locus encoding the A subunit of lactate dehydrogenase (LDH) was used to study the molecular and metabolic basis of severe hemolytic anemia due to LDH-A deficiency in the mouse. The mutant allele designated Ldh-1(a-m1Neu) codes for an enzyme that as homotetramer differs from the wild-type enzyme by a marked instability, acidic shift of the pH profile, increased K(m) for pyruvate and altered inhibition by high concentrations of this substrate. Except for the latter, all these altered properties of the mutant protein contribute to the diminished LDH activity in heterozygous and homozygous mutant individuals. Impaired energy metabolism of erythrocytes indicated by a relatively low ATP concentration is suggested to result in cell death at the end of the reticulocyte stage leading to the expression of hemolytic anemia with extreme reticulocytosis and hyperbilirubinemia. Despite the severe anemia, affected homozygous mutants exhibit approximately normal body weight and do not show noticeable impairment of viability or fertility. To date no such condition is observed in man. This discrepancy is likely due to the fact that in human erythrocytes both LDH-A and LDH-B subunits are expressed such that homozygotes for a LDH-A or LDH-B deficiency would not result in a comparably extreme LDH activity deficiency.     

Role of sph2 Gene Regulation in Hemolytic and Sphingomyelinase Activities Produced by Leptospira interrogans

Pathogenic members of the genus Leptospira are the causative agents of leptospirosis, a neglected disease of public and veterinary health concern. Leptospirosis is a systemic disease that in its severest forms leads to renal insufficiency, hepatic dysfunction, and pulmonary failure. Many strains of Leptospira produce hemolytic and sphingomyelinase activities, and a number of candidate leptospiral hemolysins have been identified based on sequence similarity to well-characterized bacterial hemolysins. Five of the putative hemolysins are sphingomyelinase paralogs. Although recombinant forms of the sphingomyelinase Sph2 and other hemolysins lyse erythrocytes, none have been demonstrated to contribute to the hemolytic activity secreted by leptospiral cells. In this study, we examined the regulation of sph2 and its relationship to hemolytic and sphingomyelinase activities produced by several L. interrogans strains cultivated under the osmotic conditions found in the mammalian host. The sph2 gene was poorly expressed when the Fiocruz L1-130 (serovar Copenhageni), 56601 (sv. Lai), and L495 (sv. Manilae) strains were cultivated in the standard culture medium EMJH. Raising EMJH osmolarity to physiological levels with sodium chloride enhanced Sph2 production in all three strains. In addition, the Pomona subtype kennewicki strain LC82-25 produced substantially greater amounts of Sph2 during standard EMJH growth than the other strains, and sph2 expression increased further by addition of salt. When 10% rat serum was present in EMJH along with the sodium chloride supplement, Sph2 production increased further in all strains. Osmotic regulation and differences in basal Sph2 production in the Manilae L495 and Pomona strains correlated with the levels of secreted hemolysin and sphingomyelinase activities. Finally, a transposon insertion in sph2 dramatically reduced hemolytic and sphingomyelinase activities during incubation of L. interrogans at physiologic osmolarity. Complementation of the mutation with the sph2 gene partially restored production of hemolytic and sphingomyelinase activities. These results indicate that the sph2 gene product contributes to the hemolytic and sphingomyelinase activities secreted by L. interrogans and most likely dominates those functions under the culture condition tested.     

A model of hemopoietic stress in a lactate dehydrogenase mouse mutant with hemolytic anemia

A codominantly inherited mutation of the lactate dehydrogenase (LDH) in the C3H mouse causes a severe hemolytic anemia in homozygous mutants, whereas viability and fertility are close to normal. Investigation of multipotent hemopoietic stem cells (CFU-S), myeloid (GM-CFC) and erythroid progenitors (BFU-E, CFU-E) in femur and spleen indicates a general shift from bone marrow to splenic hemopoiesis. In terms of total body hemopoiesis, however, the BFU-E pool is 1.4- and the CFU-E pool 19-fold enlarged, whereas CFU-S and GM-CFC show little or no deviation from normal. It is concluded that this mouse mutant is an appropriate model of long-term hemopoietic stress showing that compensation in this severe hemolytic anemia is achieved primarily by an increase of the number of the most mature erythroid progenitors.     

Characterization of zebrafish merlot/chablis as non-mammalian vertebrate models for severe congenital anemia due to protein 4.1 deficiency

The red blood cell membrane skeleton is an elaborate and organized network of structural proteins that interacts with the lipid bilayer and transmembrane proteins to maintain red blood cell morphology, membrane deformability and mechanical stability. A crucial component of red blood cell membrane skeleton is the erythroid specific protein 4.1R, which anchors the spectrin-actin based cytoskeleton to the plasma membrane. Qualitative and quantitative defects in protein 4.1R result in congenital red cell membrane disorders characterized by reduced cellular deformability and abnormal cell morphology. The zebrafish mutants merlot (mot) and chablis (cha) exhibit severe hemolytic anemia characterized by abnormal cell morphology and increased osmotic fragility. The phenotypic analysis of merlot indicates severe hemolysis of mutant red blood cells, consistent with the observed cardiomegaly, splenomegaly, elevated bilirubin levels and erythroid hyperplasia in the kidneys. The result of electron microscopic analysis demonstrates that mot red blood cells have membrane abnormalities and exhibit a severe loss of cortical membrane organization. Using positional cloning techniques and a candidate gene approach, we demonstrate that merlot and chablis are allelic and encode the zebrafish erythroid specific protein 4.1R. We show that mutant cDNAs from both alleles harbor nonsense point mutations, resulting in premature stop codons. This work presents merlot/chablis as the first characterized non-mammalian vertebrate models of hereditary anemia due to a defect in protein 4.1R integrity.     

Mutants of Burkholderia cenocepacia with a change in synthesis of N-acyl-homoserine lactones--signal molecules of Quorum Sensing regulation

By means of plasposon mutagenesis, mutants of Burkholderia cenocepacia 370 with the change in production of N-acyl-homoserine lactones (AHL), signal molecules of the Quorum Sensing system of regulation, were obtained. To localize plasposon insertions in mutant strains, fragments of chromosomal DNA containing plasposons were cloned, adjacent DNA regions sequenced, and a search for homologous nucleotide sequences in the GeneBank was initiated. It has been shown that the insertion of plasposon into gene lon encoding lon proteinase drastically decreases AHL synthesis. Upon insertion of plasposon into gene pps encoding phosphoenolpyruvate-synthase, enhancement of AHL production is observed. In mutant carrying inactivated gene lon, a strong decline of extracellular protease activity, hemolytic, and chitinolytic activities was observed in comparison with the original strain; lipase activity was not changed in this mutant. Mutation in gene pps did not affect these properties of B. cenocepacia 370. Mutations in genes lon and pps reduced the virulence of bacteria upon infection of mice.     

Essential role for TrkB receptors in hippocampus-mediated learning

Brain-derived neurotrophic factor (BDNF) and its receptor TrkB regulate both short-term synaptic functions and long-term potentiation (LTP) of brain synapses, raising the possibility that BDNF/TrkB may be involved in cognitive functions. We have generated conditionally gene targeted mice in which the knockout of the trkB gene is restricted to the forebrain and occurs only during postnatal development. Adult mutant mice show increasingly impaired learning behavior or inappropriate coping responses when facing complex and/or stressful learning paradigms but succeed in simple passive avoidance learning. Homozygous mutants show impaired LTP at CA1 hippocampal synapses. Interestingly, heterozygotes show a partial but substantial reduction of LTP but appear behaviorally normal. Thus, CA1 LTP may need to be reduced below a certain threshold before behavioral defects become apparent.     

Ras-guanine nucleotide exchange factor sos2 is dispensable for mouse growth and development

The mammalian sos1 and sos2 genes encode highly homologous members of the Son-of-sevenless family of guanine nucleotide exchange factors. They are ubiquitously expressed and play key roles in transmission of signals initiated by surface protein tyrosine kinases that are transduced into the cell through the action of membrane-associated Ras proteins. Recent reports showed that targeted disruption of the sos1 locus results in embryonic lethality. To gain insight into the in vivo function of sos2, we disrupted its catalytic CDC25-H domain by means of gene targeting techniques. Mating among heterozygous sos2(+/-) mice produced viable sos2(-/-) offspring with a normal Mendelian pattern of inheritance, indicating that the loss of sos2 does not interfere with embryo viability in the uterus. Adult homozygous mutant sos2(-/-) mice reached sexual maturity at the same age as their wild-type littermates, and both male and female null mutants were fertile. Histopathological analysis showed no observable differences between mutant and wild-type mice. Our results show that unlike the case for sos1, sos2 gene function is dispensable for normal mouse development, growth, and fertility.     

Metabolic properties of erythrocytes of normal and genetically anemic mice

The murine hemolytic anemias microcytosis (gene symbol mk), normoblastosis (nb), spherocytosis (sph), and hemolytic (ha) are inherited as autosomal recessive diseases and resemble the human hereditary hemolytic anemias caused by defective enzyme activities in erythrocytes. The activities of 14 different enzymes of the glycolytic and pentose phosphate pathways were compared in erythrocytes from normal and anemic mice, but no quantitative differences suggesting enzyme deficiency were found. There were no major changes in reduced glutathione, NAD, NADP, or methemoglobin content. The rate of entry of glucose into the glycolytic and hexose monophosphate shunt pathways of intact erythrocytes was higher in mk/mk erythrocytes than predicted. Interpretation of studies of erythrocytes from anemic mice is generally complicated by the extremely high reticulocyte and nucleated cell counts in ha/ha, sph/sph, and nb/nb mice.Investigations in Kentucky (Dr. Hutton) were supported by Research Career Development Award 1-K4-AM-70, 186-01 and NIH Research Grant AM 16013-01 from the National Institute of Arthritis and Metabolic Diseases, and those at The Jackson Laboratory (Dr. Bernstein) by NIH Research Grant HD-00254 from the National Institute of Child Health and Human Development, by U.S. Atomic Energy Commission Contract AT(30-1)-1800, and in part by the George W. Perkins Memorial Fund and by income from the Endowment Funds of The Jackson Laboratory. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

Combining Fas mutation with interleukin-2 deficiency prevents Colitis and Lupus: implicating interleukin-2 for auto-reactive T cell expansion and Fas ligand for colon epithelial cell death

Both the lpr gene defect and interleukin 2-targeted mutation (IL-2 KO) in mice are lethal. Interestingly, mice bearing both mutations live significantly longer than mice with either of the single mutant genes, approximating the life span of normal controls. They do not display the major disease phenotypes of lpr and IL-2 KO mice. Systemic autoimmune response, the accumulation of the abnormal CD4-CD8-B220+ double-negative T cells, kidney disease pathology, anemia, colon damage, and lethality are prevented. Our data indicate that IL-2 is mandatory for the expansion of auto-reactive T cells in lpr mice and that CD95 (Fas) is the critical target for the development of anemia and ulcerative colitis in IL-2 KO mice in which CD178 (FasL) on intraepithelial T cells is the major effector responsible for colon damage and lethality.     

Ablation of vitamin D signaling rescues bone, mineral, and glucose homeostasis in Fgf-23 deficient mice.

To explore further the role of the vitamin D axis for fibroblast growth factor-23 (FGF23) signaling, we mated Fgf-23 deficient (Fgf-23(-/-)) mice and vitamin D receptor (VDR) mutant mice with a non-functioning VDR. To prevent secondary hyperparathyroidism in VDR and compound mutant mice, all mice were kept on a rescue diet enriched with calcium, phosphorus, and lactose. Consistent with previous findings, Fgf-23(-/-) animals showed hypercalcemia, hyperphosphatemia, growth retardation, ectopic calcifications, severe osteoidosis, skin atrophy, and renal dysfunction. In addition, here we describe that Fgf-23(-/-) mice are hypoglycemic, and have profoundly increased peripheral insulin sensitivity and improved subcutaneous glucose tolerance, but normal renal expression of the aging suppressor gene Klotho. Although VDR and double mutants on the rescue diet still had moderately elevated parathyroid hormone serum levels and lower bone mineral density compared to wild-type mice, double mutant mice were normocalcemic and normophosphatemic, and had normal body weight, normal renal function, and no ectopic calcifications. Ablation of vitamin D signaling in compound mutants also normalized subcutaneous glucose tolerance tests and insulin secretory response. In conclusion, our results indicate that the alterations in mineral and carbohydrate metabolism present in Fgf-23(-/-) mice require an intact vitamin D signaling pathway.     

Cardiac hypertrophy in anion exchanger 1-null mutant mice with severe hemolytic anemia

Anion exchanger 1 (AE1; SLC4A1), the plasma membrane Cl(-)/HCO(3)(-) exchanger of erythrocytes, is also expressed in heart. The aim of this study was to assess the role of AE1 in heart function through study of AE1-null (AE1(-/-)) mice, which manifest severe hemolytic anemia resulting from erythrocyte fragility. Heart weight-to-body weight ratios were significantly higher in the AE1(-/-) mice than in wild-type (AE1(+/+)) littermates at both 1-3 days postnatal (3.01 +/- 0.38 vs. 1.45 +/- 0.04) and at 7 days postnatal (9.45 +/- 0.53 vs. 4.13 +/- 0.41), indicating that loss of AE1 led to cardiac hypertrophy. Heterozygous (AE1(+/-)) mice had no signs of cardiac hypertrophy. Morphology of the adult AE1(-/-) mutant heart revealed an increased left ventricular mass, accompanied by increased collagen deposition and fibrosis. M-mode echocardiography revealed dysfunction of the AE1(-/-) hearts, including dilated left ventricle end diastole and systole and expanded left ventricular mass compared with AE1(+/+) hearts. Expression of intracellular pH-regulatory mechanisms in the hypertrophic myocardium of neonate AE1(-/-) mutant mice was indistinguishable from AE1(+/-) and AE1(+/+) mice, as assessed by quantitative real-time RT-PCR. Confocal immunofluorescence revealed that, in normal mouse myocardium, AE1 is sarcolemmal, whereas AE3 and slc26a6 are found both at the sarcolemma and in internal membranes (T tubules and sarcoplasmic reticulum). These results indicate that AE1(-/-) mice, which suffer from severe hemolytic anemia and spherocytosis, display cardiac hypertrophy and impaired cardiac function, reminiscent of findings in patients with hereditary abnormalities of red blood cells. No essential role for AE1 in heart function was found.     

A mutation in the gene for delta-aminolevulinic acid dehydratase (ALAD) causes hypochromic anemia in the medaka, Oryzias latipes

A genetic screen for mutations affecting embryogenesis in the medaka, Oryzias latipes, identified a mutant, whiteout (who), that exhibited hypochromic anemia. The who mutant initially had the normal number of blood cells, but it then gradually decreased during the embryonic and larval stages. The blood cells in the who mutants show an elongated morphology and little hemoglobin activity. Genetic mapping localized who to the vicinity of a LG12 marker, olgc1. By utilizing the highly conserved synteny between medaka and pufferfish, we identified a gene for delta-aminolevulinic acid dehydratase (ALAD), which is the second enzyme in the heme synthetic pathway, as a candidate for who. We found a missense mutation in the alad gene that was tightly linked to the who phenotype, strongly suggesting that the hypochromic anemia phenotype in the who mutant is caused by a loss of the alad function. Thus, who mutants represent a model for the human disease ALAD-deficiency porphyria.     

Enzymatic defects underlying hereditary glutamate cysteine ligase deficiency are mitigated by association of the catalytic and regulatory subunits

Glutamate cysteine ligase (GCL) deficiency is a rare autosomal recessive trait that compromises production of glutathione, a critical redox buffer and enzymatic cofactor. Patients have markedly reduced levels of erythrocyte glutathione, leading to hemolytic anemia and, in some cases, impaired neurological function. Human glutamate cysteine ligase is a heterodimer comprised of a catalytic subunit (GCLC) and a regulatory subunit (GCLM), which catalyzes the initial rate-limiting step in glutathione production. Four clinical missense mutations have been identified within GCLC: Arg127Cys, Pro158Leu, His370Leu, and Pro414Leu. Here, we have evaluated the impacts of these mutations on enzymatic function in vivo and in vitro to gain further insight into the pathology. Embryonic fibroblasts from GCLC null mice were transiently transfected with wild-type or mutant GCLC, and cellular glutathione levels were determined. The four mutant transfectants each had significantly lower levels of glutathione relative to that of the wild type, with the Pro414Leu mutant being most compromised. The contributions of the regulatory subunit to GCL activity were investigated using a Saccharomyces cerevisiae model system. Mutant GCLC alone could not complement a glutathione deficient strain and required the concurrent addition of GCLM to restore growth. Kinetic characterizations of the recombinant GCLC mutants indicated that the Arg127Cys, His370Leu, and Pro414Leu mutants have compromised enzymatic activity that can largely be rescued by the addition of GCLM. Interestingly, the Pro158Leu mutant has kinetic constants comparable to those of wild-type GCLC, suggesting that heterodimer formation is needed for stability in vivo. Strategies that promote heterodimer formation and persistence would be effective therapeutics for the treatment of GCL deficiency.     

Mutants of Burkholderia cenocepacia with a change in synthesis of N-acyl-homoserine lactones—Signal molecules of quorum sensing regulation

By means of plasposon mutagenesis, mutants of Burkholderia cenocepacia 370 with the change in production of N-acyl-homoserine lactones (AHL), signal molecules of the Quorum Sensing system of regulation, were obtained. To localize plasposon insertions in mutant strains, fragments of chromosomal DNA containing plasposons were cloned, adjacent DNA regions sequenced, and a search for homologous nucleotide sequences in the GeneBank was initiated. It has been shown that the insertion of plasposon into gene lon encoding Lon proteinase drastically decreases AHL synthesis. Upon insertion of plasposon into gene pps encoding phosphoenolpyruvate-synthase, enhancement of AHL production is observed. In mutant carrying inactivated gene lon, a strong decline of extracellular protease activity, hemolytic, and chitinolytic activities was observed in comparison with the original strain; lipase activity was not changed in this mutant. Mutation in gene pps did not affect these properties of B. cenocepacia 370. Mutations in genes lon and pps reduced the virulence of bacteria upon infection of mice.     

A knock-in mouse model of congenital erythropoietic porphyria

Congenital erythropoietic porphyria (CEP) is a recessive autosomal disorder characterized by a deficiency in uroporphyrinogen III synthase (UROS), the fourth enzyme of the heme biosynthetic pathway. The severity of the disease, the lack of specific treatment except for allogeneic bone marrow transplantation, and the knowledge of the molecular lesions are strong arguments for gene therapy. An animal model of CEP has been designed to evaluate the feasibility of retroviral gene transfer in hematopoietic stem cells. We have previously demonstrated that the knockout of the Uros gene is lethal in mice (Uros(del) model). This work describes the achievement of a knock-in model, which reproduces a mutation of the UROS gene responsible for a severe UROS deficiency in humans (P248Q missense mutant). Homozygous mice display erythrodontia, moderate photosensitivity, hepatosplenomegaly, and hemolytic anemia. Uroporphyrin (99% type I isomer) accumulates in urine. Total porphyrins are increased in erythrocytes and feces, while Uros enzymatic activity is below 1% of the normal level in the different tissues analyzed. These pathological findings closely mimic the CEP disease in humans and demonstrate that the Uros(mut248) mouse represents a suitable model of the human disease for pathophysiological, pharmaceutical, and therapeutic purposes.     

A threshold requirement for Gbx2 levels in hindbrain development

Gbx2 is a homeobox gene that plays a crucial role in positioning the mid/hindbrain organizer (isthmus), which regulates midbrain and cerebellar development primarily through the secreted factor FGF8. In Gbx2 null homozygotes, rhombomeres (r) 1-3 fail to develop and the isthmic expression of Fgf8 is reduced and disorganized. These mutants fail to form a cerebellum, as it is derived from r1. Here, we analyze mice homozygous for a Gbx2 hypomorphic allele (Gbx2(neo)). Quantitative RT-PCR and RNA in situ analyses indicate that the presence of a neo-resistance cassette impairs normal Gbx2 splicing thus reducing wild-type Gbx2 mRNA levels to 6-10% of normal levels in all domains and stages examined. In Gbx2 hypomorphic mutants, gene marker and neuronal patterning analyses indicate that reduced Gbx2 expression is sufficient to support the development of r3 but not r2. The posterior region of r1, from which the lateral cerebellum develops, is unaffected in these mutants. However, the anterior region of r1 is converted to an isthmus-like tissue. Hence, instead of expressing r1 markers, this region displays robust expression of Fgf8 and Fgf17, as well as the downstream FGF targets Spry1 and Spry4. Additionally, we demonstrate that the cell division regulator cyclin D2 is downregulated, and that cellular proliferation is reduced in both the normal isthmus and in the mutant anterior r1. As a result of this transformation, the cerebellar midline fails to form. Thus, our studies demonstrate different threshold requirements for the level of Gbx2 gene product in different regions of the hindbrain.     

Role of gene 59 of bacteriophage T4 in repair of UV-irradiated and alkylated DNA in vivo.

Nonsense mutants in gene 59 (amC5, amHL628) were used to study the role of this gene in the repair of UV-damaged and alkylated DNA of bacteriophage T4 in vivo. The higher sensitivity to UV irradiation and alkylation of gene 59 mutants after exposure to these agents was established by a comparison of the survival fractions with wild type. Zonal centrifugal analysis of both parental and nascent mutant intracellular DNA molecules after UV irradiation showed that immediately after exposure the size of single-stranded DNA fragments was the same as the wild-type intracellular DNA. However, the capability of rejoining fragmented intracellular DNA was greatly reduced in the mutant. In contrast, the wild-type-infected cells under the same condition resumed DNA replication and repaired its DNA to normal size. Methyl methanesulfonate induced more randomly fragmented intracellular DNA, when compared to UV irradiation. The rate of rejoining under these conditions as judged from their sedimentation profiles was also greatly reduced in mutant-infected cells. Further evidence is presented that UV repair is not a simple consequence of arrested DNA replication, which is a phenotype of the mutant when infected in a nonpermissive host, Escherichia coli B (su minus), but rather that the DNA repair function of gene 59 is independent of the replication function. These and other data presented indicate that a product(s) of gene 59 is essential for both repair of UV lesions and repair of alkylation damage of DNA in vivo. It is suggested that gene 59 may have two functions during viral development: DNA replication and replication repair of DNA molecules.