Sickle cell disease (SCD) is one of the most common hemoglobinopathies, which is caused by the replacement of glutamic acid with valine at the sixth position of the beta-globin amino acid chain which sickling of the e...Sickle cell disease (SCD) is one of the most common hemoglobinopathies, which is caused by the replacement of glutamic acid with valine at the sixth position of the beta-globin amino acid chain which sickling of the entire red blood cells in the homozygous (Hb S/S) condition. There are many analyses and screening procedures were developed to detect sickle cell anemia in the early age of birth, especially from heel prick blood, but in case of developing countries, it would be more acceptable to detect sickle cell disorder using umbilical cord blood just after birth rather than using heel prick blood. In this study, umbilical cord blood (UCB) was used to detect β-hemoglobin gene and sickle cell disorder. Polymerase chain reaction (PCR) based analysis was done using two primers (wild-type and mutant type) to detect this disorder. A total number of 22 samples were enrolled in this experiment for PCR amplification among which nineteen samples were identified by amplification of both 267 bp and 517 bp fragments revealing heterozygous sickle cell trait (Hb A/S), whereas three samples were found to amplify of 517 bp only revealing healthy individuals. The result from PCR analysis was then collaborated with the information of the mothers of each sample to analyze the result more conveniently and found that the mothers of all individuals except the three samples had anemia or mild form of anemia, thus it was expected that the newborn might have anemia trait (Hb A/S) the exception was found in case of sample No. 9 and sample No. 15. Both samples showed the bands on 267 bp and 517 bp thus expressed the sickle cell disease trait although the mothers of these samples were not anemic. However, no samples were recorded having sickle cell anemia (9 Hb S/S). The inherent simplicity and low cost of this PCR based analysis with umbilical cord blood will be considered as an effective tool in future newborn screening in Bangladesh.展开更多
Objective To investigate whether α-hemoglobin stabilizing protein (AHSP), the α-globin-specific molecular chaperone, is regulated by erythroid transcription factor NF-E2. Methods We established the stable cell line ...Objective To investigate whether α-hemoglobin stabilizing protein (AHSP), the α-globin-specific molecular chaperone, is regulated by erythroid transcription factor NF-E2. Methods We established the stable cell line with NF-E2p45 (the larger subunit of NF-E2) short hairpin RNA to silence its expression. Western blot, real-time polymerase chain reaction, and chromatin immunoprecipitation (ChIP) analysis were performed to detect the expression of AHSP, the histone modifications at AHSP gene locus, and the binding of GATA-1 at the AHSP promoter with NF-E2p45 deficiency. ChIP was also carried out in dimethyl sulfoxide (DMSO)-induced DS19 cells and estrogen-induced G1E-ER4 cells to examine NF-E2 binding to the AHSP gene locus and its changes during cell erythroid differentiation. Finally, luciferase assay was applied in HeLa cells transfected with AHSP promoter fragments to examine AHSP promoter activity in the presence of exogenous NF-E2p45. Results We found that AHSP expression was highly dependent on NF-E2p45. NF-E2 bound to the regions across AHSP gene locus in vivo, and the transcription of AHSP was transactivated by exogenous NF-E2p45. In addition, we observed the decrease of H3K4 trimethylation and GATA-1 occupancy at the AHSP gene locus in NF-E2p45-deficient cells. Restoration of GATA-1 in G1E-ER4 cells in turn led to increased DNA binding of NF-E2p45. Conclusion NF-E2 may play an important role in AHSP gene regulation, providing new insights into the molecular mechanisms underlying the erythroid-specific expression of AHSP as well as new possibilities for β-thalassemia treatment.展开更多
文摘Sickle cell disease (SCD) is one of the most common hemoglobinopathies, which is caused by the replacement of glutamic acid with valine at the sixth position of the beta-globin amino acid chain which sickling of the entire red blood cells in the homozygous (Hb S/S) condition. There are many analyses and screening procedures were developed to detect sickle cell anemia in the early age of birth, especially from heel prick blood, but in case of developing countries, it would be more acceptable to detect sickle cell disorder using umbilical cord blood just after birth rather than using heel prick blood. In this study, umbilical cord blood (UCB) was used to detect β-hemoglobin gene and sickle cell disorder. Polymerase chain reaction (PCR) based analysis was done using two primers (wild-type and mutant type) to detect this disorder. A total number of 22 samples were enrolled in this experiment for PCR amplification among which nineteen samples were identified by amplification of both 267 bp and 517 bp fragments revealing heterozygous sickle cell trait (Hb A/S), whereas three samples were found to amplify of 517 bp only revealing healthy individuals. The result from PCR analysis was then collaborated with the information of the mothers of each sample to analyze the result more conveniently and found that the mothers of all individuals except the three samples had anemia or mild form of anemia, thus it was expected that the newborn might have anemia trait (Hb A/S) the exception was found in case of sample No. 9 and sample No. 15. Both samples showed the bands on 267 bp and 517 bp thus expressed the sickle cell disease trait although the mothers of these samples were not anemic. However, no samples were recorded having sickle cell anemia (9 Hb S/S). The inherent simplicity and low cost of this PCR based analysis with umbilical cord blood will be considered as an effective tool in future newborn screening in Bangladesh.
基金Supported by National Natural Science Foundation of China (30130026, U0632005, 30721063)National Basic Research Program of China (973 Program) (2011CB964803)+1 种基金National Laboratory of Medical Molecular Biology grant (2060204)Beijing municipal government grant (YB20081002301)
文摘Objective To investigate whether α-hemoglobin stabilizing protein (AHSP), the α-globin-specific molecular chaperone, is regulated by erythroid transcription factor NF-E2. Methods We established the stable cell line with NF-E2p45 (the larger subunit of NF-E2) short hairpin RNA to silence its expression. Western blot, real-time polymerase chain reaction, and chromatin immunoprecipitation (ChIP) analysis were performed to detect the expression of AHSP, the histone modifications at AHSP gene locus, and the binding of GATA-1 at the AHSP promoter with NF-E2p45 deficiency. ChIP was also carried out in dimethyl sulfoxide (DMSO)-induced DS19 cells and estrogen-induced G1E-ER4 cells to examine NF-E2 binding to the AHSP gene locus and its changes during cell erythroid differentiation. Finally, luciferase assay was applied in HeLa cells transfected with AHSP promoter fragments to examine AHSP promoter activity in the presence of exogenous NF-E2p45. Results We found that AHSP expression was highly dependent on NF-E2p45. NF-E2 bound to the regions across AHSP gene locus in vivo, and the transcription of AHSP was transactivated by exogenous NF-E2p45. In addition, we observed the decrease of H3K4 trimethylation and GATA-1 occupancy at the AHSP gene locus in NF-E2p45-deficient cells. Restoration of GATA-1 in G1E-ER4 cells in turn led to increased DNA binding of NF-E2p45. Conclusion NF-E2 may play an important role in AHSP gene regulation, providing new insights into the molecular mechanisms underlying the erythroid-specific expression of AHSP as well as new possibilities for β-thalassemia treatment.
