A Humanized Cooley’s Anemia Mouse Model.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1784-1784
Author(s):  
Yongliang Huo ◽  
Sean C. McConnell ◽  
Clayton L. Ulrey ◽  
Ting-Ting Zhang ◽  
Rui Yang ◽  
...  
Keyword(s):  
Blood Transfusion ◽  
Mouse Model ◽  
Adult Mouse ◽  
Globin Gene ◽  
Embryonic Stem ◽  
Fetal Hemoglobin ◽  
Globin Genes ◽  
Inherited Disorders ◽  
Nucleotide Mutation ◽  
Cooley's Anemia

Abstract Cooley’s Anemia (CA) is a heterogeneous group of inherited disorders all marked by the complete absence of adult β-globin chains in red blood cells (RBCs). Newborns with CA are healthy because of the high level of fetal hemoglobin (HbF) present at birth. As HbF levels decline during the first year of life, the absence of functional adult β-globin genes results in a severe anemia that necessitates the initiation of regular blood transfusions for the remainder of life. CA has been difficult to study in murine models due to the lack of a human fetal hemoglobin equivalent in the mouse. This study reports a novel preclinical animal model of CA that survives solely on human fetal hemoglobin at birth and is blood transfusion dependent for life upon completion of the hemoglobin switch after birth. These humanized CA mice were generated by targeted gene replacement in embryonic stem cells of the adult mouse α-globin genes with human α-globin and the adult mouse β-globin genes with a delayed switching γδβ0-globin gene cassette. The nonfunctional human β0-globin knock-in allele contains a single G to A nucleotide mutation in the first base of intervening sequence 1. Both wild-type and hereditary persistence of fetal hemoglobin (HPFH) promoter mutations were tested in the human γ-globin knock-in allele. Heterozygous knock-in mice exhibit β thalassemia intermedia. Newborn homozygous knock-in mice express 100% human hemoglobin in their RBCs, suffer from ineffective erythropoiesis, survive from one to ten days after birth, and are blood transfusion dependent for adult life. This is the first CA mouse model that recapitulates the temporal onset of the disease in human patients. This CA disease model is useful for the study of the regulation of globin gene expression, synthesis, and switching; development of transfusion and iron chelation therapies; induction of fetal hemoglobin synthesis; and the testing of novel genetic and cell-based therapies for the correction of thalassemia.

Generation of a Novel Mouse Model of Cooley’s Anemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3654-3654
Author(s):  
Yongliang Huo ◽  
Sean McConnell ◽  
Chiao-Wang Sun ◽  
Li-Chen Wu ◽  
Thomas M. Ryan
Keyword(s):  
Blood Transfusion ◽  
Mouse Model ◽  
Adult Mouse ◽  
Globin Gene ◽  
Marker Gene ◽  
Gene Replacement ◽  
Globin Genes ◽  
Splice Sites ◽  
Cooley's Anemia ◽  
Cryptic Splice Sites

Abstract A novel mouse model of Cooley’s Anemia (CA) has been generated by targeted gene replacement of the adult murine α-globin genes with human α-globin and the adult mouse β-globin genes with a human γ- to β-globin gene switching cassette containing a β0 thalassemic allele. A positive-negative gene replacement construct was designed to simultaneously delete both of the adult mouse α-globin genes by inserting a 3.8kb human α1-globin gene and a hygromycin marker gene flanked by loxP sites in murine embryonic stem (ES) cells. Both adult murine β-globin genes were deleted by insertion of an Hprt marker gene that was later replaced by a 5.6kb human Aγ-globin gene, 4.1kb human β0-globin gene, and a loxP flanked hygromycin marker gene by a “tag and exchange” strategy. The human β0-globin knock-in allele contains a single G to A nucleotide mutation in the first base of intervening sequence 1 [β0-IVS1(GtoA)-globin]. This single base change destroys the splice donor site of IVS-1 resulting in the recruitment of several cryptic splice sites. Use of these cryptic splice sites produces a frameshift in the mRNA that results in no functional β-globin polypeptide synthesis from this allele. This β0-IVS1(GtoA)-globin gene mutation is a naturally occurring β0 thalassemia allele found in Mediterranean populations. Chimeric mice were generated from both the α- and β-globin targeted cells lines. After germline transmission the α- and β-globin targeted mice were bred to cre recombinase transgenic mice to remove the marker genes. Heterozygous CA mice exhibit β thalassemia intermedia. The α- and β-globin targeted mice were interbred to produce animals homozygous for the human α1- and γβ0-globin knock-in alleles. Instead of dying early in fetal life as all current homozygous β0 thalassemia mouse models, these novel homozygous CA mice survive solely on high levels of human fetal hemoglobin (α2γ2) throughout fetal development. Newborn homozygous CA mice are blood transfusion dependent similar to β thalassemia major infants. This novel model of CA has multiple improvements over existing models of β thalassemia. Namely, CA mice express 100% human hemoglobin in their RBCs, mimic the human γ- to β-globin gene switch, synthesize no functional β-globin chains after birth, have a single mutant human β0-globin knock-in allele at each β-globin locus, and are blood transfusion dependent for life after birth.

scholarly journals Inactivation of mouse alpha-globin gene by homologous recombination: mouse model of hemoglobin H disease

Blood ◽  
1996 ◽  
Vol 88 (5) ◽  
pp. 1846-1851 ◽  
Author(s):  
J Chang ◽  
RH Lu ◽  
SM Xu ◽  
J Meneses ◽  
K Chan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mouse Model ◽  
Knockout Mice ◽  
Globin Gene ◽  
Embryonic Stem ◽  
Globin Genes ◽  
Human Homologue ◽  
Alpha Thalassemia ◽  
Alpha Globin ◽  
Hemoglobin H Disease

We have disrupted the 5′ locus of the duplicated adult alpha-globin genes by gene targeting in the mouse embryonic stem cells and created mice with alpha-thalassemia syndromes. The heterozygous knockout mice (.alpha/alpha alpha) are asymptomatic like the silent carriers in humans whereas the homozygous knockout mice (.alpha/.alpha) show hemolytic anemia. Mice with three dysfunctional alpha-globin genes generated by breeding the 5′ alpha-globin knockouts (.alpha/alpha alpha) and the deletion type alpha-thalassemia mice (../alpha alpha) produce severe hemoglobin H disease and they die in utero. These results indicate that the 5′ alpha-globin gene is the predominant locus in mice, and suggest that it is even more dominant than its human homologue.

Human Gamma Globin Gene Regulation in Knock-In Mouse Models of Anemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1779-1779
Author(s):  
Sean C. McConnell ◽  
Yongliang Huo ◽  
Shan-Run Liu ◽  
Ting-Ting Zhang ◽  
Clayton L. Ulrey ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Mouse Models ◽  
Adult Mouse ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Model Systems ◽  
Globin Genes ◽  
Human Phenotype ◽  
Globin Gene Regulation ◽  
Gene Switching

Abstract The generation of transgenic and gene targeted mouse models of human hemoglobinopathies provides valuable opportunities to test mechanisms of human globin gene regulation and experimental therapies. Yet mice do not naturally have a fetal hemoglobin, challenging our ability to adequately model the developmental onset of disease. Transgenic model systems that contain the entire human β-globin locus present obstacles to the study of human globin gene switching, including a fetal to adult globin gene switch that occurs too early in development. The generation of genetically engineered mice with a delayed human γ to β hemoglobin switch has been a major topic of interest for our laboratory. Delayed γ globin gene expression improves the clinical progression in patients as well as animal models with hemoglobinopathies. However, molecular mechanisms involved in globin gene switching are not well understood. In this study the transcriptional and epigenetic regulation of human γ to β hemoglobin switching are analyzed in novel human knock-in (KI) mouse models that complete the switch from fetal to adult hemoglobin after birth. These KI mice were generated by replacement of the adult mouse β-globin genes by homologous recombination in embryonic stem cells with a delayed switching human γ to β globin gene construct. Quantitative real-time PCR and HPLC were used to measure mouse and human embryonic, fetal, and adult globin genes through development and show that we have given the mouse a true fetal hemoglobin. Heterozygous mice express human β-like globin genes at a high level comparable to the adult mouse β globin genes. Mutations responsible for hereditary persistence of fetal hemoglobin (HPFH) in the γ globin promoter recapitulate the human phenotype in KI mice, with over 50 fold γ globin gene upregulation in adults. These HPFH KI mice also display higher γ globin levels at birth and markedly delayed γ globin gene downregulation in the weeks following birth. These studies in KI mice demonstrate that human β-like globin genes interacting with the mouse LCR are regulated in a manner similar to what is seen in humans and may be used to study the mechanisms of globin gene switching. Greater understanding of γ-globin gene regulation will be required for achieving the therapeutic goal of reactivating silenced γ-globin genes to ameliorate severe human hemoglobinopathies.

scholarly journals Inactivation of mouse alpha-globin gene by homologous recombination: mouse model of hemoglobin H disease

Blood ◽  
1996 ◽  
Vol 88 (5) ◽  
pp. 1846-1851 ◽  
Author(s):  
J Chang ◽  
RH Lu ◽  
SM Xu ◽  
J Meneses ◽  
K Chan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mouse Model ◽  
Knockout Mice ◽  
Globin Gene ◽  
Embryonic Stem ◽  
Globin Genes ◽  
Human Homologue ◽  
Alpha Thalassemia ◽  
Alpha Globin ◽  
Hemoglobin H Disease

Abstract We have disrupted the 5′ locus of the duplicated adult alpha-globin genes by gene targeting in the mouse embryonic stem cells and created mice with alpha-thalassemia syndromes. The heterozygous knockout mice (.alpha/alpha alpha) are asymptomatic like the silent carriers in humans whereas the homozygous knockout mice (.alpha/.alpha) show hemolytic anemia. Mice with three dysfunctional alpha-globin genes generated by breeding the 5′ alpha-globin knockouts (.alpha/alpha alpha) and the deletion type alpha-thalassemia mice (../alpha alpha) produce severe hemoglobin H disease and they die in utero. These results indicate that the 5′ alpha-globin gene is the predominant locus in mice, and suggest that it is even more dominant than its human homologue.

scholarly journals An HPFH Mutation in the γ-Globin Gene Promoter of Humanized Cooley's Anemia Mice Rescues Them from Perinatal Lethality and Renders Them Transfusion Independent

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1290-1290
Author(s):  
Suean Daimia Fontenard ◽  
Yongliang Huo ◽  
Shanrun Liu ◽  
Jonathan Lockhart ◽  
Michael Berlett ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Therapeutic Option ◽  
Globin Gene ◽  
Gene Promoter ◽  
Fetal Hemoglobin ◽  
Thalassemia Major ◽  
Globin Genes ◽  
Humanized Mouse Model ◽  
Perinatal Lethality ◽  
Cooley's Anemia

Abstract Cooley's Anemia (CA), β-thalassemia major, is a genetic disease caused by an impairment in β-globin protein synthesis. The resulting excess in α-globin chains causes the premature destruction of erythroid cells (ineffective erythropoiesis), anemia, and if left untreated, death within the first years of life. Several mutations in the promoters of the fetal γ-globin genes have been identified which impair the silencing of the fetal genes in adulthood, a condition termed hereditary persistence of fetal hemoglobin (HPFH). The amount of fetal hemoglobin (Hb F) expressed and the distribution within the RBC population (pancellular vs heterocellular), varies widely with different HPFH mutations. It has also been observed that thalassemia patients who co-inherit HPFH mutations that express higher levels of Hb F have milder disease symptoms. The purpose of this study is to determine whether the incorporation of a non-deletional HPFH mutation into the promoter of the human g-globin gene in a humanized mouse model of CA can rescue the animals from their perinatal lethality. Heterozygous humanized HPFH -175 mice express pancellular, high-levels of Hb F into adulthood. Homozygous HPFH -175 CA mice are rescued from their perinatal lethality, surviving solely on 100% human Hb F and are transfusion independent for life. This result is significant because it demonstrates that introduction of an HPFH mutation into the γ-globin gene promoter by gene editing may be a viable therapeutic option for CA patients in the future. Disclosures No relevant conflicts of interest to declare.

The Corfu δβ thalassemia deletion disrupts γ-globin gene silencing and reveals post-transcriptional regulation of HbF expression

Blood ◽  
2005 ◽  
Vol 105 (5) ◽  
pp. 2154-2160 ◽  
Author(s):  
Lyubomira Chakalova ◽  
Cameron S. Osborne ◽  
Yan-Feng Dai ◽  
Beatriz Goyenechea ◽  
Anna Metaxotou-Mavromati ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Gene Transcription ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Critical Threshold ◽  
Critical Element ◽  
Mrna Levels ◽  
Globin Genes ◽  
Mrna Accumulation ◽  
Post Transcriptional Regulation

Abstract The 7.2 kilobase (kb) Corfu δβ thalassemia mutation is the smallest known deletion encompassing a region upstream of the human δ gene that has been suggested to account for the vastly different phenotypes in hereditary persistence of fetal hemoglobin (HPFH) versus β thalassemia. Fetal hemoglobin (HbF) expression in Corfu heterozygotes and homozygotes is paradoxically dissimilar, suggesting conflicting theories as to the function of the region on globin gene regulation. Here, we measure γ- and β-globin gene transcription, steady-state mRNA, and hemoglobin expression levels in primary erythroid cells cultured from several patients with Corfu δβ thalassemia. We show through RNA fluorescence in situ hybridization that the Corfu deletion results in high-level transcription of the fetal γ genes in cis with a concomitant reduction in transcription of the downstream β gene. Surprisingly, we find that elevated γ gene transcription does not always result in a corresponding accumulation of γ mRNA or fetal hemoglobin, indicating a post-transcriptional regulation of γ gene expression. The data suggest that efficient γ mRNA accumulation and HbF expression are blocked until β mRNA levels fall below a critical threshold. These results explain the Corfu paradox and show that the deleted region harbors a critical element that functions in the developmentally regulated transcription of the β-globin genes.

scholarly journals Molecular analysis of Japanese delta beta-thalassemia

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1771-1776
Author(s):  
S Shiokawa ◽  
H Yamada ◽  
Y Takihara ◽  
E Matsunaga ◽  
Y Ohba ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Beta Thalassemia ◽  
Globin Genes ◽  
Beta Globin ◽  
Beta Globin Gene ◽  
Gamma Globin ◽  
Large Deletions ◽  
Deletion Junction

A DNA fragment containing the deletion junction region from a Japanese individual with homozygous delta beta-thalassemia has been cloned. A clone containing the normal DNA surrounding the 3′ breakpoint of this deletion and a clone carrying the G gamma- and A gamma-globin genes of this patient were also isolated. Sequences of the deletion junction and both gamma-globin genes were determined. A comparison of these sequences with previously determined sequences of the normal counterparts revealed that the 5′ breakpoint is located between 2,134 and 2,137 base pairs (bp) 3′ to the polyA site of the A gamma-globin gene, the 5′ breakpoint is located just downstream of the 3′ border of the fetal gamma-globin duplication unit, and no molecular defects are evident within the gamma-globin gene region. A comparison between the sequences of the normal DNA surrounding the 3′ breakpoint and the normal DNA surrounding the 5′ breakpoint shows that deletion is the result of a nonhomologous recombination event. There are A+T-rich stretches near the 5′ and 3′ breakpoints in the normal DNA, and a portion of an Aly repeat is located in the region 3′ to the 3′ breakpoint. Southern blot analysis using probes 3′ to the beta-globin gene showed that the deletion extends in the 3′ direction further than any other deletions associated with delta beta-thalassemia and hereditary persistence of fetal hemoglobin (HPFH) heretofore reported. These results are discussed in terms of the mechanism generating large deletions in mammalian cells and three models for the regulation of gamma-globin and beta-globin gene expression in humans.

scholarly journals A fetal globin gene mutation in A gamma nondeletion hereditary persistence of fetal hemoglobin increases promoter strength in a nonerythroid cell.

1988 ◽  
Vol 8 (2) ◽  
pp. 713-721 ◽  
Author(s):  
M W Rixon ◽  
R E Gelinas
Keyword(s):  
Transient Expression ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Type A ◽  
Base Substitution ◽  
Globin Genes ◽  
Wild Type ◽  
Gamma Globin ◽  
Base Substitutions ◽  
Hereditary Persistence

Single base substitutions have been identified in the promoter regions of A gamma-globin genes from individuals with certain types of nondeletion A gamma hereditary persistence of fetal hemoglobin (HPFH). The presence of these mutations is closely associated with the A gamma HPFH phenotype, but proof that they are the nondeletion HPFH determinants is lacking. To test directly whether these base substitutions can result in an increase in A gamma-globin gene transcription, we studied cosmid clones containing the G gamma- through beta-globin gene regions from individuals with Greek-type (G-to-A base substitution at -117) and Chinese-type (C-to-T base substitution at -196) A gamma HPFH in a transient expression assay. When tested as part of a cosmid clone, the Greek HPFH A gamma-globin gene consistently produced about 1.4 times as much RNA as the wild-type A gamma-globin gene when standardized against RNA transcribed from the G gamma genes in cis. The relative strengths of the normal and HPFH A gamma-globin gene promoters were also compared in transient expression assays with plasmids containing the A gamma-globin genes. Pseudo-wild-type A gamma-globin genes containing a short, transcriptionally neutral deletion were used so that two A gamma-globin genes that differed in their promoter sequences could be compared in the same transfection. The plasmid transient expression results indicated a 1.3- to 1.4-fold increase in steady-state RNA levels from the Greek-type A gamma HPFH promoter compared with the wild-type A gamma promoter, while no difference was documented between the Chinese-type A gamma HPFH promoter and the wild-type A gamma promoter.

scholarly journals An analysis of fetal hemoglobin variation in sickle cell disease: the relative contributions of the X-linked factor, beta-globin haplotypes, alpha-globin gene number, gender, and age

Blood ◽  
1995 ◽  
Vol 85 (4) ◽  
pp. 1111-1117 ◽  
Author(s):  
YC Chang ◽  
KD Smith ◽  
RD Moore ◽  
GR Serjeant ◽  
GJ Dover
Keyword(s):  
Sickle Cell ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Globin Genes ◽  
Beta Globin ◽  
Gene Number ◽  
Cohort Group ◽  
Five Factors ◽  
F Cells ◽  
Alpha Globin

Five factors have been shown to influence the 20-fold variation of fetal hemoglobin (Hb F) levels in sickle cell anemia (SS): age, sex, the alpha-globin gene number, beta-globin haplotypes, and an X-linked locus that regulates the production of Hb F-containing erythrocytes (F cells), ie, the F-cell production (FCP) locus. To determine the relative importance of these factors, we studied 257 Jamaican SS subjects from a Cohort group identified by newborn screening and from a Sib Pair study. Linear regression analyses showed that each variable, when analyzed alone, had a significant association with Hb F levels (P < .05). Multiple regression analysis, including all variables, showed that the FCP locus is the strongest predictor, accounting for 40% of Hb F variation. beta-Globin haplotypes, alpha-globin genes, and age accounted for less than 10% of the variation. The association between the beta-globin haplotypes and Hb F levels becomes apparent if the influence of the FCP locus is removed by analyzing only individuals with the same FCP phenotype. Thus, the FCP locus is the most important factor identified to date in determining Hb F levels. The variation within each FCP phenotype is modulated by factors associated with the three common beta-globin haplotypes and other as yet unidentified factor(s).

scholarly journals Concordance of a point mutation 5' to the G gamma globin gene with G gamma beta +. Hereditary persistence of fetal hemoglobin in the black population

Blood ◽  
1984 ◽  
Vol 64 (6) ◽  
pp. 1292-1296 ◽  
Author(s):  
FS Collins ◽  
CD Boehm ◽  
PG Waber ◽  
CJ Jr Stoeckert ◽  
SM Weissman ◽  
...  
Keyword(s):  
Point Mutation ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Black Population ◽  
Globin Genes ◽  
Beta Globin ◽  
Restriction Endonuclease Site ◽  
Benign Condition ◽  
The Third ◽  
Hereditary Persistence

Abstract Hereditary persistence of fetal hemoglobin (HPFH) is a genetically heterogeneous and clinically benign condition characterized by persistent expression of fetal hemoglobin (Hb F) into adulthood. In the G gamma beta + type, no major deletions in the globin gene cluster occur; adult heterozygotes produce approximately 20% Hb F, which results from overproduction of G gamma chains, with no apparent increase in production from the adjacent A gamma gene. We have recently described a point mutation 202 base pairs 5′ to the cap site of the G gamma gene in an individual with G gamma beta + HPFH. This mutation abolishes a normal ApaI restriction endonuclease site, and thus can be detected by blotting of genomic DNA. We present here further data on the ApaI mutation: (1) It occurs in six of seven families with G gamma beta + HPFH. (2) In three families, detailed haplotype analysis using 11 polymorphic restriction sites in the beta globin cluster has been done. The two that carry the missing ApaI site are identical but the third, which has a normal ApaI pattern, differs from the other two in at least two sites, one of which is a new polymorphic Nco I site between the delta and beta globin genes. This suggests the possibility of a different HPFH mutation in the third family. (3) The haplotype of the G gamma beta + HPFH chromosome carrying the ApaI mutation is different from that of 108 beta A chromosomes of black individuals that have been tested. (4) The G gamma ApaI site is normal in 61 beta A and 109 beta S alleles from non-HPFH black individuals, including 22 who share the same haplotype for the intragenic G gamma, A gamma HindIII polymorphisms. These data add support to the possibility that the -202 mutation is actually causative of the G gamma beta + HPFH phenotype.

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