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Abstract: X-linked sideroblastic anemia (XLSA) and X-linked protoporphyria (XLPP) are uncommon diseases caused by loss-of-function and gain-of-function mutations, respectively, in the erythroid form of 5-aminolevulinic acid synthetase (ALAS), ALAS2, which encodes the first enzyme in heme biosynthesis. A related congenital sideroblastic anemia (CSA) is due to mutations in SLC25A38 (solute carrier family 25 member A38), which supplies mitochondrial glycine for ALAS2 (SLC25A38-CSA). The lack of viable animal models has limited the studies on pathophysiology and development of therapies for these conditions. Here, using CRISPR-CAS9 gene editing technology, we have generated knockin mouse models that recapitulate the main features of XLSA and XLPP; and using conventional conditional gene targeting in embryonic stem cells, we also developed a faithful model of the SLC25A38-CSA. In addition to examining the phenotypes and natural history of each disease, we determine the effect of restriction or supplementation of dietary pyridoxine (vitamin B6), the essential cofactor of ALAS2, on the anemia and porphyria. In addition to the well-documented response of XLSA mutations to pyridoxine supplementation, we also demonstrate the relative insensitivity of the XLPP/EPP protoporphyrias, severe sensitivity of the XLSA models, and an extreme hypersensitivity of the SLC25A38-CSA model to pyridoxine deficiency, a phenotype that is not shared with another mouse hereditary anemia model, Hbbth3/+ β-thalassemia intermedia. Thus, in addition to generating animal models useful for examining the pathophysiology and treatment of these diseases, we have uncovered an unsuspected conditional synthetic lethality between the heme synthesis-related CSAs and pyridoxine deficiency. These findings have the potential to inform novel therapeutic paradigms for the treatment of these diseases., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Septins play key roles in mammalian cell division and cytokinesis but have not previously been implicated in a germline human disorder. A male infant with severe neutropenia and progressive dysmyelopoiesis with tetraploid myeloid precursors was identified. No known genetic etiologies for neutropenia or bone marrow failure were found. However, next-generation sequencing of germline samples from the patient revealed a novel, de novo germline stop-loss mutation in the X-linked gene SEPT6 that resulted in reduced SEPT6 staining in bone marrow granulocyte precursors and megakaryocytes. Patient skin fibroblast-derived induced pluripotent stem cells (iPSCs) produced reduced myeloid colonies, particularly of the granulocyte lineage. CRISPR/Cas9 knock-in of the patient's mutation or complete knock-out of SEPT6 was not tolerated in non-patient-derived iPSCs or human myeloid cell lines, but SEPT6 knock-out was successful in an erythroid cell line and resulting clones revealed a propensity to multinucleation. In silico analysis predicts that the mutated protein hinders the dimerization of SEPT6 coiled-coils in both parallel and antiparallel arrangements, which could in turn impair filament formation. These data demonstrate a critical role for SEPT6 in chromosomal segregation in myeloid progenitors that can account for the unusual predisposition to aneuploidy and dysmyelopoiesis., (© 2021 Wiley Periodicals LLC.)

The congenital sideroblastic anemias (CSAs) are a heterogeneous group of inherited disorders of erythropoiesis characterized by pathologic deposits of iron in the mitochondria of developing erythroblasts. Mutations in the mitochondrial glycine carrier SLC25A38 cause the most common recessive form of CSA. Nonetheless, the disease is still rare, there being fewer than 70 reported families. Here we describe the clinical phenotype and genotypes of 31 individuals from 24 families, including 11 novel mutations. We also review the spectrum of reported mutations and genotypes associated with the disease, describe the unique localization of missense mutations in transmembrane domains and account for the presence of several alleles in different populations., (© 2021 Wiley Periodicals LLC.)

Background: How specific nutrients influence adaptive immunity is of broad interest. Iron deficiency is the most common micronutrient deficiency worldwide and imparts a significant burden of global disease; however, its effects on immunity remain unclear., Methods: We used a hepcidin mimetic and several genetic models to examine the effect of low iron availability on T cells in vitro and on immune responses to vaccines and viral infection in mice. We examined humoral immunity in human patients with raised hepcidin and low serum iron caused by mutant TMPRSS6 . We tested the effect of iron supplementation on vaccination-induced humoral immunity in piglets, a natural model of iron deficiency., Findings: We show that low serum iron (hypoferremia), caused by increased hepcidin, severely impairs effector and memory responses to immunizations. The intensified metabolism of activated lymphocytes requires the support of enhanced iron acquisition, which is facilitated by IRP1/2 and TFRC. Accordingly, providing extra iron improved the response to vaccination in hypoferremic mice and piglets, while conversely, hypoferremic humans with chronically increased hepcidin have reduced concentrations of antibodies specific for certain pathogens. Imposing hypoferremia blunted the T cell, B cell, and neutralizing antibody responses to influenza virus infection in mice, allowing the virus to persist and exacerbating lung inflammation and morbidity., Conclusions: Hypoferremia, a well-conserved physiological innate response to infection, can counteract the development of adaptive immunity. This nutrient trade-off is relevant for understanding and improving immune responses to infections and vaccines in the globally common contexts of iron deficiency and inflammatory disorders., Funding: Medical Research Council, UK., Competing Interests: H.D. has sat on the advisory board of Kymab, received research funding from Pfizer and La Jolla Pharmaceutical Company, and received honoraria from Pharmacosmos and Vifor. The other authors declare no competing interests., (© 2020 The Authors.)

The congenital sideroblastic anemias (CSAs) can be caused by primary defects in mitochondrial iron-sulfur (Fe-S) cluster biogenesis. HSCB (heat shock cognate B), which encodes a mitochondrial cochaperone, also known as HSC20 (heat shock cognate protein 20), is the partner of mitochondrial heat shock protein A9 (HSPA9). Together with glutaredoxin 5 (GLRX5), HSCB and HSPA9 facilitate the transfer of nascent 2-iron, 2-sulfur clusters to recipient mitochondrial proteins. Mutations in both HSPA9 and GLRX5 have previously been associated with CSA. Therefore, we hypothesized that mutations in HSCB could also cause CSA. We screened patients with genetically undefined CSA and identified a frameshift mutation and a rare promoter variant in HSCB in a female patient with non-syndromic CSA. We found that HSCB expression was decreased in patient-derived fibroblasts and K562 erythroleukemia cells engineered to have the patient-specific promoter variant. Furthermore, gene knockdown and deletion experiments performed in K562 cells, zebrafish, and mice demonstrate that loss of HSCB results in impaired Fe-S cluster biogenesis, a defect in RBC hemoglobinization, and the development of siderocytes and more broadly perturbs hematopoiesis in vivo. These results further affirm the involvement of Fe-S cluster biogenesis in erythropoiesis and hematopoiesis and define HSCB as a CSA gene.

SF3B1 is recurrently mutated in chronic lymphocytic leukemia (CLL), but its role in the pathogenesis of CLL remains elusive. Here, we show that conditional expression of Sf3b1-K700E mutation in mouse B cells disrupts pre-mRNA splicing, alters cell development, and induces a state of cellular senescence. Combination with Atm deletion leads to the overcoming of cellular senescence and the development of CLL-like disease in elderly mice. These CLL-like cells show genome instability and dysregulation of multiple CLL-associated cellular processes, including deregulated B cell receptor signaling, which we also identified in human CLL cases. Notably, human CLLs harboring SF3B1 mutations exhibit altered response to BTK inhibition. Our murine model of CLL thus provides insights into human CLL disease mechanisms and treatment., (Copyright © 2018 Elsevier Inc. All rights reserved.)

YARS2 variants have previously been described in patients with myopathy, lactic acidosis and sideroblastic anemia 2 (MLASA2). YARS2 encodes the mitochondrial tyrosyl-tRNA synthetase, which is responsible for conjugating tyrosine to its cognate mt-tRNA for mitochondrial protein synthesis. Here we describe 14 individuals from 11 families presenting with sideroblastic anemia and YARS2 variants that we identified using a sideroblastic anemia gene panel or exome sequencing. The phenotype of these patients ranged from MLASA to isolated congenital sideroblastic anemia. As in previous cases, inter- and intra-familial phenotypic variability was observed, however, this report includes the first cases with isolated sideroblastic anemia and patients with biallelic YARS2 variants that have no clinically ascertainable phenotype. We identified ten novel YARS2 variants and three previously reported variants. In vitro amino-acylation assays of five novel missense variants showed that three had less effect on the catalytic activity of YARS2 than the most commonly reported variant, p.(Phe52Leu), associated with MLASA2, which may explain the milder phenotypes in patients with these variants. However, the other two missense variants had a more severe effect on YARS2 catalytic efficiency. Several patients carried the common YARS2 c.572 G>T, p.(Gly191Val) variant (minor allele frequency =0.1259) in trans with a rare deleterious YARS2 variant. We have previously shown that the p.(Gly191Val) variant reduces YARS2 catalytic activity. Consequently, we suggest that biallelic YARS2 variants, including severe loss-of-function alleles in trans of the common p.(Gly191Val) variant, should be considered as a cause of isolated congenital sideroblastic anemia, as well as the MLASA syndromic phenotype., (Copyright© 2018 Ferrata Storti Foundation.)

Unlike primary myelofibrosis (PMF) in adults, myelofibrosis in children is rare. Congenital (inherited) forms of myelofibrosis (cMF) have been described, but the underlying genetic mechanisms remain elusive. Here we describe 4 families with autosomal recessive inherited macrothrombocytopenia with focal myelofibrosis due to germ line loss-of-function mutations in the megakaryocyte-specific immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor G6b-B ( G6b , C6orf25 , or MPIG6B ). Patients presented with a mild-to-moderate bleeding diathesis, macrothrombocytopenia, anemia, leukocytosis and atypical megakaryocytes associated with a distinctive, focal, perimegakaryocytic pattern of bone marrow fibrosis. In addition to identifying the responsible gene, the description of G6b-B as the mutated protein potentially implicates aberrant G6b-B megakaryocytic signaling and activation in the pathogenesis of myelofibrosis. Targeted insertion of human G6b in mice rescued the knockout phenotype and a copy number effect of human G6b-B expression was observed. Homozygous knockin mice expressed 25% of human G6b-B and exhibited a marginal reduction in platelet count and mild alterations in platelet function; these phenotypes were more severe in heterozygous mice that expressed only 12% of human G6b-B. This study establishes G6b-B as a critical regulator of platelet homeostasis in humans and mice. In addition, the humanized G6b mouse will provide an invaluable tool for further investigating the physiological functions of human G6b-B as well as testing the efficacy of drugs targeting this receptor., (© 2018 by The American Society of Hematology.)

During terminal differentiation, the global protein complement is remodeled, as epitomized by erythrocytes, whose cytosol is ~98% globin. The erythroid proteome undergoes a rapid transition at the reticulocyte stage; however, the mechanisms driving programmed elimination of preexisting cytosolic proteins are unclear. We found that a mutation in the murine Ube2o gene, which encodes a ubiquitin-conjugating enzyme induced during erythropoiesis, results in anemia. Proteomic analysis suggested that UBE2O is a broad-spectrum ubiquitinating enzyme that remodels the erythroid proteome. In particular, ribosome elimination, a hallmark of reticulocyte differentiation, was defective in Ube2o -/- mutants. UBE2O recognized ribosomal proteins and other substrates directly, targeting them to proteasomes for degradation. Thus, in reticulocytes, the induction of ubiquitinating factors may drive the transition from a complex to a simple proteome., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Symptomatic β-thalassemia is one of the globally most common inherited disorders. The initial clinical presentation is variable. Although common hematological analyses are typically sufficient to diagnose the disease, sometimes the diagnosis can be more challenging. We describe a series of patients with β-thalassemia whose diagnosis was delayed, required bone marrow examination in one affected member of each family, and revealed ringed sideroblasts, highlighting the association of this morphological finding with these disorders. Thus, in the absence of characteristic congenital sideroblastic mutations or causes of acquired sideroblastic anemia, the presence of ringed sideroblasts should raise the suspicion of β-thalassemia., (© 2016 Wiley Periodicals, Inc.)

The congenital sideroblastic anemias (CSAs) are a heterogeneous group of inherited blood disorders characterized by pathological mitochondrial iron deposition in erythroid precursors. Each known cause has been attributed to a mutation in a protein associated with heme biosynthesis, iron-sulfur cluster biogenesis, mitochondrial translation, or a component of the mitochondrial respiratory chain. Here, we describe a recurring mutation, c.276_278del, p.F93del, in NDUFB11, a mitochondrial respiratory complex I-associated protein encoded on the X chromosome, in 5 males with a variably syndromic, normocytic CSA. The p.F93del mutation results in respiratory insufficiency and loss of complex I stability and activity in patient-derived fibroblasts. Targeted introduction of this allele into K562 erythroleukemia cells results in a proliferation defect with minimal effect on erythroid differentiation potential, suggesting the mechanism of anemia in this disorder., (© 2016 by The American Society of Hematology.)

More than 80% of patients with the refractory anemia with ring sideroblasts subtype of myelodysplastic syndrome (MDS) have mutations in Splicing Factor 3B, Subunit 1 (SF3B1). We generated a conditional knockin mouse model of the most common SF3B1 mutation, Sf3b1(K700E). Sf3b1(K700E) mice develop macrocytic anemia due to a terminal erythroid maturation defect, erythroid dysplasia, and long-term hematopoietic stem cell (LT-HSC) expansion. Sf3b1(K700E) myeloid progenitors and SF3B1-mutant MDS patient samples demonstrate aberrant 3' splice-site selection associated with increased nonsense-mediated decay. Tet2 loss cooperates with Sf3b1(K700E) to cause a more severe erythroid and LT-HSC phenotype. Furthermore, the spliceosome modulator, E7017, selectively kills SF3B1(K700E)-expressing cells. Thus, SF3B1(K700E) expression reflects the phenotype of the mutation in MDS and may be a therapeutic target in MDS., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Mitochondrial myopathy with lactic acidosis and sideroblastic anemia (MLASA) is an oxidative phosphorylation disorder, with primary clinical manifestations of myopathic exercise intolerance and a macrocytic sideroblastic anemia. One cause of MLASA is recessive mutations in PUS1, which encodes pseudouridine (Ψ) synthase 1 (Pus1p). Here we describe a mouse model of MLASA due to mutations in PUS1. As expected, certain Ψ modifications were missing in cytoplasmic and mitochondrial tRNAs from Pus1(-/-) animals. Pus1(-/-) mice were born at the expected Mendelian frequency and were non-dysmorphic. At 14 weeks the mutants displayed reduced exercise capacity. Examination of tibialis anterior (TA) muscle morphology and histochemistry demonstrated an increase in the cross sectional area and proportion of myosin heavy chain (MHC) IIB and low succinate dehydrogenase (SDH) expressing myofibers, without a change in the size of MHC IIA positive or high SDH myofibers. Cytochrome c oxidase activity was significantly reduced in extracts from red gastrocnemius muscle from Pus1(-/-) mice. Transmission electron microscopy on red gastrocnemius muscle demonstrated that Pus1(-/-) mice also had lower intermyofibrillar mitochondrial density and smaller mitochondria. Collectively, these results suggest that alterations in muscle metabolism related to mitochondrial content and oxidative capacity may account for the reduced exercise capacity in Pus1(-/-) mice.

Pathogenic variants in mitochondrial aminoacyl-tRNA synthetases result in a broad range of mitochondrial respiratory chain disorders despite their shared role in mitochondrial protein synthesis. LARS2 encodes the mitochondrial leucyl-tRNA synthetase, which attaches leucine to its cognate tRNA. Sequence variants in LARS2 have previously been associated with Perrault syndrome, characterized by premature ovarian failure and hearing loss (OMIM #615300). In this study, we report variants in LARS2 that are associated with a severe multisystem metabolic disorder. The proband was born prematurely with severe lactic acidosis, hydrops, and sideroblastic anemia. She had multisystem complications with hyaline membrane disease, impaired cardiac function, a coagulopathy, pulmonary hypertension, and progressive renal disease and succumbed at 5 days of age. Whole exome sequencing of patient DNA revealed compound heterozygous variants in LARS2 (c.1289C>T; p.Ala430Val and c.1565C>A; p.Thr522Asn). The c.1565C>A (p.Thr522Asn) LARS2 variant has previously been associated with Perrault syndrome and both identified variants are predicted to be damaging (SIFT, PolyPhen). Muscle and liver samples from the proband did not display marked mitochondrial respiratory chain enzyme deficiency. Immunoblotting of patient muscle and liver showed LARS2 levels were reduced in liver and complex I protein levels were reduced in patient muscle and liver. Aminoacylation assays revealed p.Ala430Val LARS2 had an 18-fold loss of catalytic efficiency and p.Thr522Asn a 9-fold loss compared to wild-type LARS2. We suggest that the identified LARS2 variants are responsible for the severe multisystem clinical phenotype seen in this baby and that mutations in LARS2 can result in variable phenotypes.

The congenital sideroblastic anemias (CSAs) are relatively uncommon diseases characterized by defects in mitochondrial heme synthesis, iron-sulfur (Fe-S) cluster biogenesis, or protein synthesis. Here we demonstrate that mutations in HSPA9, a mitochondrial HSP70 homolog located in the chromosome 5q deletion syndrome 5q33 critical deletion interval and involved in mitochondrial Fe-S biogenesis, result in CSA inherited as an autosomal recessive trait. In a fraction of patients with just 1 severe loss-of-function allele, expression of the clinical phenotype is associated with a common coding single nucleotide polymorphism in trans that correlates with reduced messenger RNA expression and results in a pseudodominant pattern of inheritance., (© 2015 by The American Society of Hematology.)

Iron is essential for numerous cellular processes. For diagnostic purposes iron-related parameters in patients are assessed by clinical chemical blood analysis including the analysis of ferritin, transferrin and iron levels. Here, we retrospectively evaluated the use of these parameters in the phenotype-driven Munich N-ethyl-N-nitrosourea mouse mutagenesis project for the generation of novel animal models for human diseases. The clinical chemical blood analysis was carried out on more than 10,700 G1 and G3 offspring of chemically mutagenized inbred C3H mice to detect dominant and recessive mutations leading to deviations in the plasma levels of iron-related plasma parameters. We identified animals consistently exhibiting altered plasma ferritin or transferrin values. Transmission of the phenotypic deviations to the subsequent generations led to the successful establishment of three mutant lines with increased plasma ferritin levels. For two of these lines the causative mutations were identified in the Fth1gene and the Ireb2 gene, respectively. Thus, novel mouse models for the functional analysis of iron homeostasis were established by a phenotype-driven screen for mutant mice.

Mutations in genes encoding proteins that are involved in mitochondrial heme synthesis, iron-sulfur cluster biogenesis, and mitochondrial protein synthesis have previously been implicated in the pathogenesis of the congenital sideroblastic anemias (CSAs). We recently described a syndromic form of CSA associated with B-cell immunodeficiency, periodic fevers, and developmental delay (SIFD). Here we demonstrate that SIFD is caused by biallelic mutations in TRNT1, the gene encoding the CCA-adding enzyme essential for maturation of both nuclear and mitochondrial transfer RNAs. Using budding yeast lacking the TRNT1 homolog, CCA1, we confirm that the patient-associated TRNT1 mutations result in partial loss of function of TRNT1 and lead to metabolic defects in both the mitochondria and cytosol, which can account for the phenotypic pleiotropy.

X-linked sideroblastic anemia (XLSA) is the most common form of congenital sideroblastic anemia. In affected males, it is uniformly associated with partial loss-of-function missense mutations in the erythroid-specific heme biosynthesis protein 5-aminolevulinate synthase 2 (ALAS2). Here, we report five families with XLSA owing to mutations in a GATA transcription factor binding site located in a transcriptional enhancer element in intron 1 of the ALAS2 gene. As such, this study defines a new class of mutations that should be evaluated in patients undergoing genetic testing for a suspected diagnosis of XLSA., (Copyright © 2013 Wiley Periodicals, Inc.)

Mutagenesis screens can establish mouse models of utility for the study of critical biological processes such as iron metabolism. Such screens can produce mutations in novel genes or establish novel alleles of known genes, both of which can be useful tools for study. In order to identify genes of relevance to hematologic as well as other phenotypes, we performed N-ethyl-N-nitrosourea mutagenesis in C57BL/6J mice. An anemic mouse was identified and a putative mutation was characterized by mapping, sequencing and in vitro activity analysis. The mouse strain was backcrossed for ten generations then phenotypically characterized with respect to a previously established null mouse strain. Potential modifying loci were identified by quantitative trait locus analysis. Mapping and sequencing in an anemic mouse termed hem8 identified an I286F substitution in Tmprss6, a serine protease essential for iron metabolism; this substitution impaired in vitro protease activity. After backcrossing to C57BL6/J for ten generations, the hem8(-/-) strain exhibited a phenotype similar in some but not all aspects to that of Tmprss6(-/-) mice. The hem8 and Tmprss6-null mutations were allelic. Both hem8(-/-) and Tmprss6(-/-) mice responded similarly to pharmacological modulators of bone morphogenetic protein signaling, a key regulator of iron metabolism. Quantitative trait locus analysis in the hem8 strain identified potential modifying loci on chromosomes 2, 4, 7 and 10. In conclusion, the hem8 mouse model carries a novel allele of Tmprss6. Potential uses for this strain in the study of iron metabolism are discussed.

Defects in the availability of haem substrates or the catalytic activity of the terminal enzyme in haem biosynthesis, ferrochelatase (Fech), impair haem synthesis and thus cause human congenital anaemias. The interdependent functions of regulators of mitochondrial homeostasis and enzymes responsible for haem synthesis are largely unknown. To investigate this we used zebrafish genetic screens and cloned mitochondrial ATPase inhibitory factor 1 (atpif1) from a zebrafish mutant with profound anaemia, pinotage (pnt (tq209)). Here we describe a direct mechanism establishing that Atpif1 regulates the catalytic efficiency of vertebrate Fech to synthesize haem. The loss of Atpif1 impairs haemoglobin synthesis in zebrafish, mouse and human haematopoietic models as a consequence of diminished Fech activity and elevated mitochondrial pH. To understand the relationship between mitochondrial pH, redox potential, [2Fe-2S] clusters and Fech activity, we used genetic complementation studies of Fech constructs with or without [2Fe-2S] clusters in pnt, as well as pharmacological agents modulating mitochondrial pH and redox potential. The presence of [2Fe-2S] cluster renders vertebrate Fech vulnerable to perturbations in Atpif1-regulated mitochondrial pH and redox potential. Therefore, Atpif1 deficiency reduces the efficiency of vertebrate Fech to synthesize haem, resulting in anaemia. The identification of mitochondrial Atpif1 as a regulator of haem synthesis advances our understanding of the mechanisms regulating mitochondrial haem homeostasis and red blood cell development. An ATPIF1 deficiency may contribute to important human diseases, such as congenital sideroblastic anaemias and mitochondriopathies.

Red blood cells are essential for oxygen transport and other physiologic processes. Red cell characteristics are typically determined by complete blood counts which measure parameters such as hemoglobin levels and mean corpuscular volumes; these parameters reflect the quality and quantity of red cells in the circulation at any particular moment. To identify the genetic determinants of red cell parameters, we performed genome-wide association analysis on LG/J×SM/J F2 and F34 advanced intercross lines using single nucleotide polymorphism genotyping and a novel algorithm for mapping in the combined populations. We identified significant quantitative trait loci for red cell parameters on chromosomes 6, 7, 8, 10, 12, and 17; our use of advanced intercross lines reduced the quantitative trait loci interval width from 1.6- to 9.4-fold. Using the genomic sequences of LG/J and SM/J mice, we identified nonsynonymous coding single nucleotide polymorphisms in candidate genes residing within quantitative trait loci and performed sequence alignments and molecular modeling to gauge the potential impact of amino acid substitutions. These results should aid in the identification of genes critical for red cell physiology and metabolism and demonstrate the utility of advanced intercross lines in uncovering genetic determinants of inherited traits.

Microcephaly affects approximately 1% of the population and is associated with mental retardation, motor defects and, in some cases, seizures. We analyzed the mechanisms underlying brain size determination in a mouse model of human microcephaly. The Hertwig's anemia (an) mutant shows peripheral blood cytopenias, spontaneous aneuploidy and a predisposition to hematopoietic tumors. We found that the an mutation is a genomic inversion of exon 4 of Cdk5rap2, resulting in an in-frame deletion of exon 4 from the mRNA. The finding that CDK5RAP2 human mutations cause microcephaly prompted further analysis of Cdk5rap2(an/an) mice and we demonstrated that these mice exhibit microcephaly comparable to that of the human disease, resulting from striking neurogenic defects that include proliferative and survival defects in neuronal progenitors. Cdk5rap2(an/an) neuronal precursors exit the cell cycle prematurely and many undergo apoptosis. These defects are associated with impaired mitotic progression coupled with abnormal mitotic spindle pole number and mitotic orientation. Our findings suggest that the reduction in brain size observed in humans with mutations in CDK5RAP2 is associated with impaired centrosomal function and with changes in mitotic spindle orientation during progenitor proliferation.

Five spontaneous, allelic mutations in the alpha-spectrin gene, Spna1, have been identified in mice (spherocytosis [sph], sph(1J), sph(2J), sph(2BC), sph(Dem)). All cause severe hemolytic anemia. Here, analysis of 3 new alleles reveals previously unknown consequences of red blood cell (RBC) spectrin deficiency. In sph(3J), a missense mutation (H2012Y) in repeat 19 introduces a cryptic splice site resulting in premature termination of translation. In sph(Ihj), a premature stop codon occurs (Q1853Stop) in repeat 18. Both mutations result in markedly reduced RBC membrane spectrin content, decreased band 3, and absent beta-adducin. Reevaluation of available, previously described sph alleles reveals band 3 and adducin deficiency as well. In sph(4J), a missense mutation occurs in the C-terminal EF hand domain (C2384Y). Notably, an equally severe hemolytic anemia occurs despite minimally decreased membrane spectrin with normal band 3 levels and present, although reduced, beta-adducin. The severity of anemia in sph(4J) indicates that the highly conserved cysteine residue at the C-terminus of alpha-spectrin participates in interactions critical to membrane stability. The data reinforce the notion that a membrane bridge in addition to the classic protein 4.1-p55-glycophorin C linkage exists at the RBC junctional complex that involves interactions between spectrin, adducin, and band 3.

Background: Sideroblastic anemias are heterogeneous congenital and acquired bone marrow disorders characterized by pathologic iron deposits in mitochondria of erythroid precursors. Among the congenital sideroblastic anemias (CSAs), the most common form is X-linked sideroblastic anemia, due to mutations in 5-aminolevulinate synthase (ALAS2). A novel autosomal recessive CSA, caused by mutations in the erythroid specific mitochondrial transporter SLC25A38, was recently defined. Other known etiologies include mutations in genes encoding the thiamine transporter SLC19A2, the RNA-modifying enzyme pseudouridine synthase 1 (PUS1), a mitochondrial ATP-binding cassette transporter (ABCB7), glutaredoxin 5 (GLRX5), as well as mitochondrial DNA deletions. Despite these known diverse causes, in a substantial portion of CSA cases a presumed genetic defect remains unknown., Procedure: In the context of the recent discovery of SLC25A38 as a major novel cause, we systematically analyzed a large cohort of previously unreported CSA patients. Sixty CSA probands (28 females, 32 males) were examined for ALAS2, SLC25A38, PUS1, GLRX5, and ABCB7 mutations. SLC19A2 and mitochondrial DNA were only analyzed if characteristic syndromic features were apparent., Results: Twelve probands had biallelic mutations in SLC25A38. Seven ALAS2 mutations were detected in eight sporadic CSA cases, two being novel. We also identified a novel homozygous null PUS1 mutation and novel mitochondrial DNA deletions in two patients with Pearson syndrome. No mutations were encountered in GLRX5, ABCB7, or SLC19A2., Conclusions: The remaining undefined probands (43%) can be grouped according to gender, family, and clinical characteristics, suggesting novel X-linked and autosomal recessive forms of CSA., ((c) 2009 Wiley-Liss, Inc.)

The sideroblastic anemias are a heterogeneous group of congenital and acquired hematological disorders whose morphological hallmark is the presence of ringed sideroblasts--bone marrow erythroid precursors containing pathologic iron deposits within mitochondria. Here, by positional cloning, we define a previously unknown form of autosomal recessive nonsyndromic congenital sideroblastic anemia, associated with mutations in the gene encoding the erythroid specific mitochondrial carrier family protein SLC25A38, and demonstrate that SLC25A38 is important for the biosynthesis of heme in eukaryotes.

Hereditary forms of iron-deficiency anemia, including animal models, have taught us much about the normal physiologic control of iron metabolism. However, the discovery of new informative mutants is limited by the natural mutation frequency. To address this limitation, we have developed a screen for heritable abnormalities of red blood cell morphology in mice with single-nucleotide changes induced by the chemical mutagen ethylnitrosourea (ENU). We now describe the first strain, fragile-red, with hypochromic microcytic anemia resulting from a Y228H substitution in the ferrireductase Steap3 (Steap3(Y288H)). Analysis of the Steap3(Y288H) mutant identifies a conserved motif required for targeting Steap3 to internal compartments and highlights how phenotypic screens linked to mutagenesis can identify new functional variants in erythropoiesis and ascribe function to previously unidentified motifs.

Mouse models have proven invaluable for understanding erythropoiesis. Here, we describe an autosomal recessive, inherited anemia in the mouse mutant hem6. Hematologic and transplantation analyses reveal a mild, congenital, hypochromic, microcytic anemia intrinsic to the hematopoietic system that is associated with a decreased red blood cell zinc protoporphyrin to heme ratio, indicative of porphyrin insufficiency. Intercross matings show that hem6 can suppress the porphyric phenotype of mice with erythropoietic protoporphyria (EPP). Furthermore, iron uptake studies in hem6 reticulocytes demonstrate defective incorporation of iron into heme that can be partially corrected by the addition of porphyrin precursors. Gene expression and enzymatic assays indicate that erythroid 5-aminolevulinic acid synthase (Alas2) is decreased in hem6 animals, suggesting a mechanism that could account for the anemia. Overall, these data lead to the hypothesis that hem6 encodes a protein that directly or indirectly regulates the expression of Alas2.

Iron deficiency is usually attributed to chronic blood loss or inadequate dietary intake. Here, we show that iron deficiency anemia refractory to oral iron therapy can be caused by germline mutations in TMPRSS6, which encodes a type II transmembrane serine protease produced by the liver that regulates the expression of the systemic iron regulatory hormone hepcidin. These findings demonstrate that TMPRSS6 is essential for normal systemic iron homeostasis in humans.

Primary ciliary dyskinesia (PCD) results from ciliary dysfunction and is commonly characterized by sinusitis, male infertility, hydrocephalus, and situs inversus. Mice homozygous for the nm1054 mutation develop phenotypes associated with PCD. On certain genetic backgrounds, homozygous mutants die perinatally from severe hydrocephalus, while mice on other backgrounds have an accumulation of mucus in the sinus cavity and male infertility. Mutant sperm lack mature flagella, while respiratory epithelial cilia are present but beat at a slower frequency than wild-type cilia. Transgenic rescue demonstrates that the PCD in nm1054 mutants results from the loss of a single gene encoding the novel primary ciliary dyskinesia protein 1 (Pcdp1). The Pcdp1 gene is expressed in spermatogenic cells and motile ciliated epithelial cells. Immunohistochemistry shows that Pcdp1 protein localizes to sperm flagella and the cilia of respiratory epithelial cells and brain ependymal cells in both mice and humans. This study demonstrates that Pcdp1 plays an important role in ciliary and flagellar biogenesis and motility, making the nm1054 mutant a useful model for studying the molecular genetics and pathogenesis of PCD.

We identified a family with gray platelet syndrome (GPS) segregating as a sex-linked trait. Affected males had a mild bleeding disorder, thrombocytopenia, and large agranular platelets characteristic of GPS, while obligate carrier females were asymptomatic but had dimorphic platelets on peripheral smear. Associated findings included mild erythrocyte abnormalities in affected males. Linkage analysis revealed a 63 cM region on the X chromosome between markers G10578 and DXS6797, which segregated with the platelet phenotype and included the GATA1 gene. Sequencing of GATA1 revealed a G-to-A mutation at position 759 corresponding to amino acid change Arg216Gln. This mutation was previously described as a cause of X-linked thrombocytopenia with thalassemia (XLTT) but not of gray platelet syndrome. Our findings suggest that XLTT is within a spectrum of disorders constituting the gray platelet syndrome, and we propose that GATA1 is an upstream regulator of the genes required for platelet alpha-granule biogenesis.

X-linked sideroblastic anemia with ataxia (XLSA/A) is a rare syndromic form of inherited sideroblastic anemia associated with spinocerebellar ataxia, and is due to mutations in the mitochondrial ATP-binding cassette transporter Abcb7. Here, we show that Abcb7 is essential for hematopoiesis and formally demonstrate that XLSA/A is due to partial loss of function mutations in Abcb7 that directly or indirectly inhibit heme biosynthesis.

Proper fatty acid metabolism is critical for hair and skin development and maintenance. The acyl-CoA binding protein (Acbp) is a widely expressed protein that binds long-chain fatty acyl-CoA esters and plays a role in fatty acyl-CoA transport and pool formation. However, loss of function of Acbp in the whole animal has not been investigated. Here, we show that deletion of Acbp in mouse results in sebocyte hyperplasia and sparse, matted hair with a greasy appearance. Consistent with these gross abnormalities, Acbp is highly expressed in the pilosebaceous units of mouse skin as determined by Northern analysis and in situ hybridization. Loss of Acbp also results in fatty acid metabolism abnormalities, with hair lipid profiles showing altered levels of triacylglycerols and nearly co-migrating lipids. These data suggest that Acbp plays a role in triacylglycerol biosynthesis, and that regulation of this process is important for proper hair and skin development and maintenance in the mouse.

Iron and copper are essential for all organisms, assuming critical roles as cofactors in many enzymes. In eukaryotes, the transmembrane transport of these elements is a highly regulated process facilitated by the single electron reduction of each metal. Previously, we identified a mammalian ferrireductase, Steap3, critical for erythroid iron homeostasis. Now, through homology, expression, and functional studies, we characterize all 4 members of this protein family and demonstrate that 3 of them, Steap2, Steap3, and Steap4, are not only ferrireductases but also cupric reductases that stimulate cellular uptake of both iron and copper in vitro. Finally, the pattern of tissue expression and subcellular localization of these proteins suggest they are physiologically relevant cupric reductases and ferrireductases in vivo.

Proteins with iron-sulfur (Fe-S) clusters participate in multiple metabolic pathways throughout the cell. The mitochondrial ABC half-transporter Abcb7, which is mutated in X-linked sideroblastic anemia with ataxia in humans, is a functional ortholog of yeast Atm1p and is predicted to export a mitochondrially derived metabolite required for cytosolic Fe-S cluster assembly. Using an inducible Cre/loxP system to delete exons 9 and 10 of the Abcb7 gene, we examined the phenotype of mice deficient in Abcb7. We found that Abcb7 was essential in extra-embryonic tissues early in gestation and that the mutant allele exhibits an X-linked parent-of-origin lethality effect. Furthermore, using X-chromosome inactivation assays and tissue-specific deletions, Abcb7 was found to be essential for the development and function of numerous other cell types and tissues. A notable exception to this was liver, where loss of Abcb7 impaired cytosolic Fe-S cluster assembly but was not lethal. In this situation, control of iron regulatory protein 1, a key cytosolic modulator of iron metabolism, which is responsive to the availability of cytosolic Fe-S clusters, was impaired and contributed to the dysregulation of hepatocyte iron metabolism. Altogether, these studies demonstrate the essential nature of Abcb7 in mammals and further substantiate a central role for mitochondria in the biogenesis of cytosolic Fe-S proteins.

Hypochromic, microcytic anemias are typically the result of inadequate hemoglobin production because of globin defects or iron deficiency. Here, we describe the phenotypic characteristics and pathogenesis of a new recessive, hypochromic, microcytic anemia mouse mutant, nm1054. Although the mutation nm1054 is pleiotropic, also resulting in sparse hair, male infertility, failure to thrive, and hydrocephaly, the anemia is the focus of this study. Hematologic analysis reveals a moderately severe, congenital, hypochromic, microcytic anemia, with an elevated red cell zinc protoporphyrin, consistent with functional erythroid iron deficiency. However, serum and tissue iron analyses show that nm1054 animals are not systemically iron deficient. From hematopoietic stem cell transplantation and iron uptake studies in nm1054 reticulocytes, we provide evidence that the nm1054 anemia is due to an intrinsic hematopoietic defect resulting in inefficient transferrin-dependent iron uptake by erythroid precursors. Linkage studies demonstrate that nm1054 maps to a genetic locus not previously implicated in microcytic anemia or iron phenotypes.

The reduction of iron is an essential step in the transferrin (Tf) cycle, which is the dominant pathway for iron uptake by red blood cell precursors. A deficiency in iron acquisition by red blood cells leads to hypochromic, microcytic anemia. Using a positional cloning strategy, we identified a gene, six-transmembrane epithelial antigen of the prostate 3 (Steap3), responsible for the iron deficiency anemia in the mouse mutant nm1054. Steap3 is expressed highly in hematopoietic tissues, colocalizes with the Tf cycle endosome and facilitates Tf-bound iron uptake. Steap3 shares homology with F(420)H(2):NADP(+) oxidoreductases found in archaea and bacteria, as well as with the yeast FRE family of metalloreductases. Overexpression of Steap3 stimulates the reduction of iron, and mice lacking Steap3 are deficient in erythroid ferrireductase activity. Taken together, these findings indicate that Steap3 is an endosomal ferrireductase required for efficient Tf-dependent iron uptake in erythroid cells.

We have studied the flexed-tail (f) mouse to gain insight into mammalian mitochondrial iron metabolism. Flexed-tail animals have axial skeletal abnormalities and a transient embryonic and neonatal anemia characterized by pathologic intramitochondrial iron deposits in erythrocytes. Mitochondrial iron accumulation is the hallmark of sideroblastic anemias, which typically result from defects in heme biosynthesis or other pathways that lead to abnormal erythroid mitochondrial iron utilization. To clone the f gene, we used positional cloning techniques, and identified a frameshift mutation in a mitochondrial transmembrane protein. The mutated gene, Sfxn1, is the prototype of a novel family of evolutionarily conserved proteins present in eukaryotes.

Hypotransferrinemic (Trf(hpx/hpx)) mice have a severe deficiency in serum transferrin (Trf) as the result of a spontaneous mutation linked to the murine Trf locus. They are born alive, but before weaning, die from severe anemia if they are not treated with exogenous Trf or red blood cell transfusions. We have determined the molecular basis of the hpx mutation. It results from a single point mutation, which alters an invariable nucleotide in the splice donor site after exon 16 of the Trf gene. No normal Trf messenger RNA (mRNA) is made from the hpx allele. A small amount of mRNA results from the usage of cryptic splice sites within exon 16. The predominant cryptic splice site produces a Trf mRNA carrying a 27-base pair (bp), in-frame deletion. Less than 1% of normal levels of a Trf-like protein is found in the serum of Trf(hpx/hpx) mice, most likely resulting from translation of the internally deleted mRNA. Despite their severe Trf deficiency, however, Trf(hpx/hpx) mice initially treated with transferrin injections can survive after weaning without any further treatment. They have massive tissue iron overload develop in all nonhematopoietic tissues, while they continue to have severe iron deficiency anemia. Their liver iron burden is 100-fold greater than that of wild-type mice and 15- to 20-fold more than that of mice lacking the hemochromatosis gene, Hfe. Trf(hpx/hpx) mice thus provide an additional model with a defined molecular defect for the study of genetic iron disorders.