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PubMed2003

Evaluation of optimal expression cassette in retrovirus vector for beta-thalassemia gene therapy.

Trials of retroviral vector-mediated human beta-globin gene transfer were hampered by low titers, unstable vector transmission, and low-level expression of transferred gene. With the goal of optimizing the retrovirally encoded human beta-globin gene expression cassette for gene therapy of beta-thalassemia, we generated 3 series of vector constructs (a total of 12 constructs) and investigated the effects of the proximal promoter, 3' - enhancer, and derivatives from the beta-locus control region or alpha-major regulatory element on virus titer, vector transmission stability, and gene expression. The virus titers for 9 of the 12 vector constructs ranged between 2.8 x 10(4) cfu/mL and 1.0 x 10(6) cfu/mL. We found that proviral DNA was intact in most G418- resistant murine erythroleukemia (MEL) cell clones for 5 vector constructs, while obvious genetic instability was observed for 4 other vector constructs. MEL cells harboring the intact provirus were induced to differentiate, and human beta-globin gene expression was analyzed with RNase protection assay. The percentage of human beta-globin transcript relative to endogenous murine alpha-globin transcript were 101.8 +/- 64.3% (n = 10), 40.1 +/- 28.7% (n = 4), 31.1 +/- 31.9% (n = 12), 52.4 +/- 11.2% (n = 12), and 53.6 +/- 8.6% (n = 12) for the 5 constructs, respectively, demonstrating the development of optimized retroviral vectors for beta-globin gene therapy with murine erythroid cell lines as a model. Unexpectedly, we also documented that the point mutation 8700(C-->T) in DNase I hypersensitive site 2 (HS2) core fragment might contribute to low-level expression of the human beta-globin gene, based on a comparison of results from transfected and transduced MEL cells and sequence analysis of proviral DNA.

Animals↗
PubMed2001

Gene transfer into nonhuman primate hematopoietic stem cells: implications for gene therapy.

Hematopoietic stem cells (HSCs) are desirable targets for gene therapy because of their self-renewal and multilineage differentiation abilities. Retroviral vectors are extensively used for HSC gene therapy. However, the initial human trials of HSC gene marking and therapy showed that the gene transfer efficiency into human HSCs with retroviral vectors was very low in contrast to the much higher efficiency observed in murine experiments. The more quiescent nature of human HSCs and the lower density of retroviral receptors on them hindered the efficient gene transfer with retroviral vectors. Since nonhuman primates have marked similarity to humans in all aspects including the HSC biology, their models are considered to be important to evaluate and improve gene transfer into human HSCs. Using these models, clinically relevant levels (around 10% or even more) of gene-modified cells in peripheral blood have recently been achieved after gene transfer into HSCs and their autologous transplantation. This has been made possible by improving ex vivo transduction conditions such as introduction of Flt-3 ligand and specific fibronectin fragment (CH-296) into ex vivo culture during transduction, and the use of retroviral vectors pseudotyped with the gibbon ape leukemia virus or feline endogenous retrovirus envelope. Other strategies including the use of lentiviral vectors and in vivo selective expansion of gene-modified cells with the drug resistance gene or selective amplifier gene (also designated the molecular growth switch) are now being tested to further increase the fraction of gene-modified cells using nonhuman primate models. In addition to the high gene transfer efficiency, high-level and long-term expression of transgenes in human HSCs and their progeny is also required for effective HSC gene therapy. For this purpose, other backbones of retroviral vectors such as the murine stem cell virus and cis-DNA elements, such as the ss-globin locus control region and the chromatin insulator, also need to be tested in nonhuman primate models. Nonhuman primate studies will continue to provide an important framework for human HSC gene therapy. Well-designed nonhuman primate studies will also offer unique insights into the HSCs, immune system, and transplantation biology characteristic of large animals.

Animals↗
PubMed2005

Association of polymorphisms and haplotypes in the human growth hormone 1 (GH1) gene with breast cancer.

The growth hormone 1 (GH1)/insulin-like growth factor I (IGF-I) axis plays an important role in the development of breast cancer. By binding to its receptor, GH1 stimulates the production of IGF-I and its binding protein IGFBP3, resulting in the regulation of cell proliferation, differentiation and apoptosis. The GH1 gene expression is regulated by a highly polymorphic proximal promoter and a distal locus control region (LCR) 14.5 kb upstream of the gene. We investigated the effect of single nucleotide polymorphisms (SNPs) in the LCR and in the promoter region and an intron 4 SNP (IVS4+90 T/A) on breast cancer risk in a large cohort of Polish and German familial breast cancer cases and controls. SNPs in the LCR did not show an influence on breast cancer risk, either alone or in haplotypes. Three SNPs in the promoter region (G-340T, A-68G/C and A-63T/C) showed an increased and four SNPs (A-137G, G-119T, G-93delG and T-4G) a decreased allele frequency in the cases compared with the controls. Two of the SNPs (A-137G and G-93delG) lead to a decreased breast cancer risk among the minor allele carriers in the joint analysis of the two populations (odds ratio (OR) 0.62, 95% confidence interval (95% CI) 0.44-0.89, P = 0.01 and OR 0.65, 95% CI 0.47-0.90, P = 0.01, respectively). Haplotype analysis with these seven promoter SNPs revealed a protective association (OR 0.61, 95% CI 0.37-1.00, P = 0.04) for the haplotype GAGdAAT, containing the G-93delG variant allele, which in the single analysis already showed a protective effect. The effect was marginally stronger in combination with the LCR GC haplotype (OR 0.49, 95% CI 0.23-1.01, P = 0.04).

Adult↗
PubMed1992

Normal delta-globin gene sequences in Sardinian nondeletional delta beta-thalassemia.

In order to clarify the reasons for the reduced Hb A2 levels in Sardinian delta beta-thalassemia, we characterized, both by cloning and sequence analysis and by direct sequencing of amplified DNA, the delta-globin gene from an individual of Sardinian descent who is a compound heterozygote for the beta zero-thalassemia codon 39 (C-->T) nonsense mutation and the Sardinian delta beta-thalassemia [codon 39(C-->T)/-196(C-->T)A gamma]. The analysis of the delta-globin gene from the delta beta-thalassemia chromosome revealed an entirely normal sequence. The defective function of the delta-globin gene in this determinant is thus likely related to a suppressive effect of the in cis nondeletional high persistence of fetal hemoglobin mutation of the A gamma gene, probably resulting from an increased capability of the relative promoter to interact with the locus control region.

Base Sequence↗
PubMed1995

Structural characterization of the beta-globin gene cluster in an individual expressing a very low level of G gamma globin chains.

We report the case of a normal individual displaying an extremely unbalanced G gamma/A gamma-globin ratio (G gamma-globin chains undetectable by urea/triton/ acrylamide gel electrophoresis and just reaching the threshold of detection by high performance liquid chromatography) associated with a very low level of G gamma-globin mRNA (at the most 5% of total gamma-mRNA after reverse transcriptase polymerase chain reaction determination). By DNA Southern blotting and sequencing, the very low level of G gamma-globin chains in this individual was found in association with subhaplotype [+ -----] (Hinc II 5' to epsilon, Xmn I 5' to G gamma, Hind III in G gamma and A gamma, Hinc II in and 3' to psi beta), with G gamma- and A gamma-globin gene sequences of the B type chromosome, and with a number of AT repeats in the locus control region hypersensitive site-2 site, similar to that reported to be associated with the Bantu beta S haplotype. These structural characteristics, described for the first time combined in the same individual, suggest that the G gamma/A gamma ratio in adults, is controlled by sequences distributed all along the beta-globin gene cluster.

Adolescent↗
PubMedDate not supplied

Expression of a bovine GABAA receptor alpha1-subunit cDNA in murine erythroleukaemia cells.

A plasmid vector has been constructed by insertion of the cDNA encoding the alpha1 subunit of the bovine GABAA receptor into the LCR/MEL expression vector pNV1 downstream of the human globin locus control region between the promoter and the second intron of the beta-globin gene to produce pNVGABAalpha. This plasmid was transfected into murine erythroleukemia (MEL) cells using electroporation to obtain recombinant cells. Parental and recombinant cells were tested by both RNA dot blot and electrophysiological analysis for the presence of bovine GABAA receptor alpha1 subunit mRNA. Parental MEL cells did not express GABA-gated chloride channels but recombinant cells were sensitive to pressure-applied GABA. The GABA responses reversed at the equilibrium potential predicted for chloride ions. These results show that the alpha1 subunit of the bovine GABAA receptor inserts in the plasma membrane of the MEL cells and forms homo-oligomeric chloride channels that are gated by GABA. Our studies suggest, therefore, that the LCR/MEL system can be used for the expression of neurotransmitter receptor genes.

Animals↗
PubMed2001

A CD2-green fluorescence protein-transgenic mouse reveals very late antigen-4-dependent CD8+ lymphocyte rolling in inflamed venules.

Intravital microscopy allows detailed analysis of leukocyte trafficking in vivo, but fails to identify the nature of leukocytes investigated. Here, we describe the development of a CD2-enhanced green fluorescence protein (EGFP)-transgenic mouse to characterize lymphocyte trafficking during inflammation in vivo. A CD2-EGFP plasmid construct including the CD2 promoter, the EGFP transgene, and the CD2 locus control region was injected into B6CBA/F1 pronuclei. EGFP+ offspring were backcrossed into C57BL/6 mice for six generations. Flow cytometry demonstrated that all peripheral blood EGFP+ cells were positive for CD2 and negative for the granulocyte Ag Ly 6-G (GR-1). EGFP(high) cells stained positive for CD2, CD3, CD8, TCR beta-chain, and NK1.1 but did not express the B cell and monocyte markers CD45RA, CD19, and CD11b. In vitro stimulation assays revealed no difference in lymphocyte proliferation and IL-2 secretion between EGFP+ and EGFP- mice. Intravital microscopy of untreated or TNF-alpha-treated cremaster muscle venules showed EGFP+ cells in vivo, but these cells did not roll or adhere to the vessel wall. In cremaster muscle venules treated with both TNF-alpha and IFN-gamma, EGFP(high) cells rolled, adhered, and transmigrated at a rolling velocity slightly higher (11 microm/s) than that of neutrophils (10 microm/s). Blocking alpha4 integrin with a mAb increased rolling velocity to 24 microm/s. These findings show that CD8+ T cells roll in TNF-alpha/IFN-gamma-pretreated vessels in vivo via an alpha4 integrin-dependent pathway.

Animals↗
PubMed2002

Activation of CD8 T cells by antigen expressed in the pituitary gland.

Ag expressed exclusively in the anterior pituitary gland and secreted locally by pituitary somatotrophs can gain access to the MHC class I presentation pathway and activate CD8 T cells. Influenza nucleoprotein (NP) was expressed as a transgene under the control of the human growth hormone (GH) locus control region. Activation of monoclonal F5 CD8 T cells specific for NP resulted in spontaneous autoimmune pathology of the pituitary gland in mice transgenic for both NP and the F5 TCR. Destruction of somatotrophs resulted in drastically reduced GH levels in adult mice and a dwarf phenotype. Adoptive transfer of F5 T cells into NP-transgenic hosts resulted in full T cell activation, first demonstrable in regional lymph nodes, followed by their migration to the pituitary gland. Despite the presence of activated, IFN-gamma-producing CD8 T cells in the pituitary gland and a slight reduction in pituitary GH levels, no effect on growth was observed. Thus, CD8 T cells have access to the neuroendocrine system and get fully activated in the absence of CD4 help, but Ag recognition in this location causes autoimmune pathology only in the presence of excessive CD8 T cell numbers.

Adoptive Transfer↗
PubMed2003

Expression of a constitutively active Stat6 in vivo alters lymphocyte homeostasis with distinct effects in T and B cells.

IL-4 is a critical cytokine in the regulation of immune responses and genesis of atopy. Engagement of the IL-4R activates multiple signaling pathways, including the transcription factor Stat6. Stat6-deficient mice demonstrate the importance of this factor in lymphocyte proliferation, gene expression, and Th cell differentiation. Recently, a mutant Stat6 (Stat6VT) was generated that is transcriptionally active independent of IL-4 stimulation. To determine the ability of a constitutively active Stat6 to mimic IL-4-stimulated responses, we have generated transgenic mice expressing Stat6VT under control of the CD2 locus control region, restricting expression to lymphoid populations. The phenotype of Stat6VT transgenic mice is similar, but not identical, to IL-4 transgenic mice, suggesting a critical role for Stat6-independent signaling pathways in the generation of some IL-4 responses in vivo. The expression of a constitutively active Stat6 in vivo increases surface expression of IL-4-induced genes and increases serum levels of IgG1 and IgE, compared with nontransgenic mice. Stat6VT expression increases Th2 differentiation in vivo and in vitro. Stat6VT expression also dramatically alters homeostasis of peripheral lymphocyte populations resulting in decreased CD3(+) cells and increased B220(+) cells, compared with nontransgenic littermates. Altered T and B cell populations correlate with an activated phenotype and increased cell death in transgenic T cell, but not B cell, populations. Together these results suggest that expression of a constitutively active Stat6 has distinct effects on B and T lymphocytes.

Animals↗
PubMed2004

An important role of Nrf2-ARE pathway in the cellular defense mechanism.

The antioxidant responsive element (ARE) is a cis-acting regulatory element of genes encoding phase II detoxification enzymes and antioxidant proteins, such as NAD(P)H: quinone oxidoreductase 1, glutathione S-transferases, and glutamate-cysteine ligase. Interestingly, it has been reported that Nrf2 (NF-E2-related factor 2) regulates a wide array of ARE-driven genes in various cell types. Nrf2 is a basic leucine zipper transcription factor, which was originally identified as a binding protein of locus control region of beta-globin gene. The DNA binding sequence of Nrf2 and ARE sequence are very similar, and many studies demonstrated that Nrf2 binds to the ARE sites leading to up-regulation of downstream genes. The function of Nrf2 and its downstream target genes suggests that the Nrf2-ARE pathway is important in the cellular antioxidant defense system. In support of this, many studies showed a critical role of Nrf2 in cellular protection and anti-carcinogenicity, implying that the Nrf2-ARE pathway may serve as a therapeutic target for neurodegenerative diseases and cancers, in which oxidative stress is closely implicated.

Animals↗
PubMed1999

Deficient transcription of mouse mast cell protease 4 gene in mutant mice of mi/mi genotype.

The mi locus encodes a member of the basic-helix-loop-helix-leucine zipper (bHLH-Zip) protein family of transcription factors (hereafter called MITF). We reported that expression of the mouse mast cell protease 5 (MMCP-5) and MMCP-6 genes were deficient in cultured mast cells (CMC) derived from mutant mice of mi/mi genotype. Despite the reduced expression of both MMCP-5 and MMCP-6, their regulation mechanisms were different. Because MMCP-5 is a chymase and MMCP-6 a tryptase, there was a possibility that the difference in regulation mechanisms was associated with their different characteristics as proteases. We compared the regulation mechanisms of another chymase, MMCP-4, with those of MMCP-5 and MMCP-6. The expression of the MMCP-4 gene was also deficient in mi/mi CMC. The overexpression of the normal (+) MITF but not of mi-MITF normalized the poor expression of the MMCP-4 gene in mi/mi CMC, indicating the involvement of +-MITF in transactivation of the MMCP-4 gene. Although MMCP-4 is chymase as MMCP-5, the regulation of MMCP-4 expression was more similar to MMCP-6 than to MMCP-5. We also showed the deficient expression of granzyme B and cathepsin G genes in mi/mi CMC. Genes encoding granzyme B, cathepsin G, MMCP-4, and MMCP-5 are located on chromosome 14. Because all these genes showed deficient expression in mi/mi CMC, there is a possibility that MITF might regulate the expression of these genes through a locus control region.

Animals↗
PubMed1999

The evolution of trichromatic color vision by opsin gene duplication in New World and Old World primates.

Trichromacy in all Old World primates is dependent on separate X-linked MW and LW opsin genes that are organized into a head-to-tail tandem array flanked on the upstream side by a locus control region (LCR). The 5' regions of these two genes show homology for only the first 236 bp, although within this region, the differences are conserved in humans, chimpanzees, and two species of cercopithecoid monkeys. In contrast, most New World primates have only a single polymorphic X-linked opsin gene; all males are dichromats and trichromacy is achieved only in those females that possess a different form of this gene on each X chromosome. By sequencing the upstream region of this gene in a New World monkey, the marmoset, we have been able to demonstrate the presence of an LCR in an equivalent position to that in Old World primates. Moreover, the marmoset sequence shows extensive homology from the coding region to the LCR with the upstream sequence of the human LW gene, a distance of >3 kb, whereas homology with the human MW gene is again limited to the first 236 bp, indicating that the divergent MW sequence identifies the site of insertion of the duplicated gene. This is further supported by the presence of an incomplete Alu element on the upstream side of this insertion point in the MW gene of both humans and a cercopithecoid monkey, with additional Alu elements present further upstream. Therefore, these Alu elements may have been involved in the initial gene duplication and may also be responsible for the high frequency of gene loss and gene duplication within the opsin gene array. Full trichromacy is present in one species of New World monkey, the howler monkey, in which separate MW and LW genes are again present. In contrast to the separate genes in humans, however, the upstream sequences of the two howler genes show homology with the marmoset for at least 600 bp, which is well beyond the point of divergence of the human MW and LW genes, and each sequence is associated with a different LCR, indicating that the duplication in the howler monkey involved the entire upstream region. [The sequence data described in this paper have been submitted to GenBank under accession nos. AF155218, AF156715, and AF156716.]

Animals↗
PubMed1999

Far upstream regulatory elements enhance position-independent and uterus-specific expression of the murine alpha1(I) collagen promoter in transgenic mice.

The stage- and tissue-specific expression of many eukaryotic genes is regulated by cis-regulatory elements, some of which are located in proximity to the start site of transcription whereas others have been identified at considerable distances. In previous studies we have identified far upstream DNase I-hypersensitive sites in the murine alpha1(I) collagen (Col1a1) gene, which may play a role in the regulation of this abundantly expressed gene. Here we have cloned several of these sites into reporter gene constructs containing the Col1a1 promoter driving the green fluorescent protein (GFP) reporter gene and tested their possible functions in transfection experiments and transgenic mice. In transient and stable transfections none of the hypersensitive sites had a significant effect on Col1a1 promoter activity, indicating that they do not contain a classical transcriptional enhancer. In transgenic animals one element located at -18 to -19.5 kb enhanced the position-independent activity of the linked Col1a1 promoter and may be part of a locus control region. Another element located at -7 to -8 kb specifically enhanced reporter gene expression in the uteri of transgenic mice, suggesting that it contains a novel transcriptional enhancer that may be involved in the regulation of type I collagen expression in tissue remodeling in the uterus during the estrous cycle. Our studies also demonstrate the versatility of the GFP reporter gene for use in transgenic animals because it can be analyzed in live animals, whole mount embryos, histological thin sections, or primary cell cultures, and it can be quantified very sensitively in tissue or cell extracts using a fluorometer.

Animals↗
PubMed2002

[The possible mechanisms underlying low expression of human beta-globin gene cloned in a retroviral vector].

Murine erythroleukemia (MEL) cell line was used as a model to evaluate the potential value of retroviral construct containing human beta-globin express io n cassette in gene therapy of beta-thalassemia and to explore possible mechanisms underlying low expression of retrovirally cloned human beta-globin gene. A recombinant retroviral vector was constructed, which harbored 2.0 kb beta-globin gene w it h a 374 bp deletion in intervening sequence II coupled with a mini locus control region (miniLCR) composed of DNaseI hypersensitive sites (HS) 2 and 3 from human beta-LCR. The recombinant retroviruses were generated from an established psi-2 producer cell line, and by transient transfection of amphotropic packaging cell l ine psi-A, respectively. The integrity of provirus in transduced MEL cells was determined using Southern blot, and the expression of transferred human beta-globin gene was detected using RNase protection assay. The structure of provirus was further analyzed by sequence analysis of PCR products from genomic DNA of MEL individual clone as template. The results demonstrated that the average expression of human beta-globin gene was (52.4-/+11.2)% (n=12) and (73.8-/+14.3)% (n=12, without copy-number determination), compared with that of endogenous murine alpha-globin ge ne, in MEL cells transduced with the recombinant retrovirus from transient transfection of psi-A and MEL cells transfected with the construct, respectively. In M EL cells transduced with virus from psi-2 producer cell line, however, the average expression was less than 3%. A point mutation was detected in HS2 of provirus i n MEL cell clone with low expression of human beta-globin gene. The possible mechanisms involved in low expression, including position effect, DNA methylation a nd RNA interference are discussed in addition to the point mutation.

Base Sequence↗
PubMedDate not supplied

[Genome domains and domain regulatory elements].

Gene activity regulation at the level of genome domains is considered with special emphasis on locus control regions (LCR), which are thought to act as dominant regulatory elements providing for the active state of tissue-specific gene domains. Detailed analysis was made of experimental data on the organization and function of mammalian beta-globin gene LCR. On recent evidence, this LCR is necessary for high-level expression of the globin genes, but not for the active state of the domain. These data are compared with earlier findings suggesting a key role of LCR in the organization of active genome domains. The boundaries of functional genome domains are also considered.

Animals↗
PubMed2003

CP2 binding to the promoter is essential for the enhanced transcription of globin genes in erythroid cells.

We have previously reported that the reduced level of CP2 suppresses the mouse alpha- and beta-globin gene expression and hemoglobin synthesis during terminal differentiation of mouse erythroleukemia (MEL) cells in vitro [Chae et al. (1999)]. As an extension of this study, we demonstrated that human alpha-, epsilon-, and gamma- globin genes were also suppressed by the reduced expression of CP2 in K562 cells. To address how much CP2 contributes in the regulation of globin gene expression, we measured transcriptional activities of the wild type alpha-globin promoter and its various factor-binding sites mutants in erythroid and nonerythroid cells. Interestingly, CP2 site dependent transcriptional activation occurred in an erythroid-cell specific manner, even though CP2 is ubiquitously expressed. In addition, CP2 site mutation within the alpha-promoter severely suppressed promoter activity in differentiated, but not in undifferentiated MEL cells, suggesting that the CP2 binding site is needed for the enhanced transcription of globin genes during erythroid differentiation. When the human beta-globin locus control region was linked to the alpha-promoter, suppression was more severe in the CP2 site mutant in differentiated MEL cells. Overall data indicate that CP2 is a major factor in the regulation of globin expression in human and mouse erythroid cells, and CP2 binding to the globin gene promoter is essential for the enhanced transcription of globin genes in erythroid differentiation.

Animals↗
PubMed1992

Cell-specific expression of high levels of human S100 beta in transgenic mouse brain is dependent on gene dosage.

The beta-subunit of S100 protein (S100 beta) is highly conserved in the mammalian brain. The gene coding for human S100 beta has been mapped to chromosome 21. In order to study the consequences of overexpression of the S100 beta gene, transgenic mice were generated by microinjection of a 17.3 kilobase human genomic fragment containing the three exons and the transcription control elements of the human S100 beta gene. Mice from four transgenic lines carried approximately 10-100 transgene copies. Northern blotting demonstrated a tissue-specific and gene dose-dependent expression of human S100 beta mRNA in mouse brain. Increased expression of S100 beta mRNA was correlated with an increased production of S100 beta protein. Examination of brain sections by in situ hybridization and immunocytochemistry indicated that S100 beta was localized globally to astrocytes, as well as to discrete neurons in the mesencephalic and motor trigeminal, facial, and lemniscus nuclei in both normal and transgenic mice. In peripheral tissues, human S100 beta was expressed at 10-50-fold lower levels than in brain. The strict gene dosage dependence and cell specificity of transgene expression suggest the presence of a locus control region (LCR) in the human S100 beta gene. The mice tolerated 10-100-fold higher than normal levels of S100 beta gene expression in brain without any gross physical or behavioral abnormalities. The high-level expression and cell specificity of the S100 beta promoter/LCR suggest that it may provide a valuable tool to direct the expression of other transgenic products to specific cell types in the CNS.

Animals↗
PubMed2004

Dominant influence of gamma-globin promoter polymorphisms on fetal haemoglobin expression in sickle cell disease.

Polymorphisms of multiple cis-acting elements in the beta-globin locus are associated with variable fetal haemoglobin (HbF) level in sickle cell disease. We developed a multiplex assay permitting simultaneous analysis of three polymorphic cis elements spanning 53 kb of the beta-globin locus. We identified concordance between polymorphic alleles in gamma- and beta-globin promoters however a significant number of betaS-chromosomes were identified with polymorphisms in hypersensitive site 2 (HS2) of the beta-globin locus control region juxtaposed to atypical cis alleles in the gamma-promoter. Analysis of an unusually large number of such hybrid haplotype chromosomes provided unique insight into HbF level associated with specific cis alleles. Associations between cis alleles and HbF level in patients were verified by in vitro functional analysis. Our findings indicate that compared to HS2, polymorphism in the gamma-promoter exerts a dominant influence on HbF level in sickle cell disease.

Alleles↗