Use of biotinylated probes for detecting sickle cell anemia.

1985 ◽  
Vol 31 (7) ◽  
pp. 1203-1206 ◽  
Author(s):  
G J Garbutt ◽  
J T Wilson ◽  
G S Schuster ◽  
J J Leary ◽  
D C Ward
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Genomic Dna ◽  
Globin Gene ◽  
Blot Hybridization ◽  
Beta Globin ◽  
Radioactive Materials ◽  
Dna Fragment ◽  
Cell Gene ◽  
Biotinylated Probes

Abstract Earlier, we reported that the 5' end of the normal beta-globin gene (beta) resides on a 1.14-kilobase DNA fragment, whereas the 5' end of the sickle cell gene (beta s) resides on a 1.34-kilobase fragment. In that blot hybridization analysis, we used genomic DNA digested with restricted endonuclease Mst II, and radioactive probes with short half-life. We demonstrate here that, if a biotinylated probe is used instead in a slightly modified procedure, sickle cell anemia can be quickly and directly detected if there is as much as 5 micrograms of total genomic DNA in the sample. This procedure obviates the special precautions necessary when radioactive materials are used.

scholarly journals Haplotypes of the beta-globin gene as prognostic factors in sickle-cell disease

1999 ◽  
Vol 5 (6) ◽  
pp. 1254-1258
Author(s):  
M. A. El Hazmi ◽  
A. S. Warsy ◽  
N. Bashir ◽  
A. Beshlawi ◽  
I. R. Hussain
Keyword(s):  
Saudi Arabia ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Clinical Presentation ◽  
Globin Gene ◽  
Beta Globin ◽  
Cell Disease ◽  
Beta Globin Gene ◽  
The North ◽  
Cell Gene

Wecollaborated with researchers from Egypt, Syrian Arab Republic and Jordan in a study of patients with sickle-cell disease from those countries, and from various parts of Saudi Arabia, in order to investigate the influence of genetics on the clinical presentation of the disease, and to attempt to determine the origin of the sickle-cell gene in Arabs. Our results suggest that beta-globin gene haplotypes influence the clinical presentation of sickle-cell disease, and that there are at least two major foci for the origin of the sickle-cell gene, one in the eastern part of Saudi Arabia, and the other in the populations of North Africa and the north-western part of the Arabian peninsula

Nonisotopic M13 probes for detecting the beta-globin gene: application to diagnosis of sickle cell anemia.

1987 ◽  
Vol 33 (8) ◽  
pp. 1368-1371 ◽  
Author(s):  
E Sheldon ◽  
D E Kellogg ◽  
C Levenson ◽  
W Bloch ◽  
L Aldwin ◽  
...  
Keyword(s):  
Horseradish Peroxidase ◽  
Sickle Cell ◽  
Restriction Fragment ◽  
Globin Gene ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Beta Globin ◽  
Beta Globin Gene ◽  
Human Dna ◽  
Peroxidase Conjugate

Abstract M13 DNA probes labeled with biotinylated psoralen and a streptavidin-horseradish peroxidase conjugate provide nonradioactive detection of the sickle cell and normal alleles of the beta-globin locus. The two biotinylated probes contain single-stranded sequences complementary to two different Sau3AI restriction fragments from the 5' region of the beta-globin gene and double-stranded M13 vector sequences. These probes are labeled with biotinylated psoralen photochemically linked to DNA. After hybridization, the presence of biotinylated probe bound to target DNA is detected in 3 h by using a streptavidin-horseradish peroxidase conjugate and the substrate, 3,3',5,5'-tetramethylbenzidine. Digestion of the normal (beta A) allele of the beta-globin gene with MstII (or isoschizomers) yields a 1.14-kb restriction fragment, while digestion of the mutant beta S allele yields a 1.34-kb fragment. These fragments can be resolved by gel electrophoresis and detected by Southern blot hybridization. The nonradioisotopic probe system can detect the beta-globin restriction fragment in as little as 0.5 microgram of human DNA and can distinguish heterozygotes (beta A beta S) from homozygotes (beta A beta A or beta S beta S) in 2.0 micrograms of human DNA.

scholarly journals Phytomedicines and Nutraceuticals: Alternative Therapeutics for Sickle Cell Anemia

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Ngozi Awa Imaga
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Globin Gene ◽  
Alternative Therapy ◽  
Clinical Manifestations ◽  
Base Substitution ◽  
Beta Globin ◽  
Inherited Disease ◽  
Gene Encoding

Sickle cell anemia is a genetically inherited disease in which the “SS” individual possesses an abnormal beta globin gene. A single base substitution in the gene encoding the humanβ-globin subunit results in replacement ofβ6 glutamic acid by valine, leading to the devastating clinical manifestations of sickle cell disease. This substitution causes drastic reduction in the solubility of sickle cell hemoglobin (HbS) when deoxygenated. Under these conditions, the HbS molecules polymerize to form long crystalline intracellular mass of fibers which are responsible for the deformation of the biconcave disc shaped erythrocyte into a sickle shape. First-line clinical management of sickle cell anemia include, use of hydroxyurea, folic acid, amino acids supplementation, penicillinprophylaxis, and antimalarial prophylaxis to manage the condition and blood transfusions to stabilize the patient's hemoglobin level. These are quite expensive and have attendant risk factors. However, a bright ray of hope involving research into antisickling properties of medicinal plants has been rewarding. This alternative therapy using phytomedicines has proven to not only reduce crisis but also reverse sickling (in vitro). The immense benefits of phytomedicines and nutraceuticals used in the management of sickle cell anemia are discussed in this paper.

Influence of Globin Gene Modifiers on Baseline and Hydroxyurea-Induced Fetal Hemoglobin Levels in Children with Sickle Cell Anemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3171-3171
Author(s):  
Russell E. Ware ◽  
Barry Eggleston ◽  
Tatiana Abramova ◽  
Sherri A. Zimmerman ◽  
Alice Lail ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Globin Genes ◽  
Beta Globin ◽  
Gene Number ◽  
Gamma Globin ◽  
Alpha Globin ◽  
Hydroxyurea Therapy

Abstract Fetal hemoglobin (HbF) is recognized as a major determinant of clinical disease severity in children and adults with sickle cell anemia (SCA). Patients with elevated HbF levels have a milder disease course, and many current therapeutic protocols for SCA include pharmacological induction of HbF. However, baseline and treatment HbF levels vary widely due to presumed genetic and environmental factors. Recognized globin gene modifiers of HbF include the beta globin haplotype and a potential contribution from concomitant alpha thalassemia. To characterize more fully the influence of globin gene modifiers on both baseline and treatment HbF levels, we retrospectively determined the beta globin haplotype (Benin, CAR, Senegal, Cameroon, or Arab-Indian) by selective gamma globin gene nucleotide sequencing and the alpha globin gene number (2, 3, or 4) by PCR for 67 African-American children with SCA receiving hydroxyurea therapy at stable maximal tolerated dose (MTD). The four beta globin haplotypes and frequencies identified in our cohort of children include Benin (0.61), CAR (0.17), Senegal (0.12), and Cameroon (0.10). The number of alpha globin genes and frequencies identified were 4 genes (0.72), 3 genes (0.25) and 2 genes (0.03). Baseline and MTD HbF levels were analyzed according to each variable. The average baseline HbF value for the entire cohort of children was 7.7 ± 4.4% (median 7.6%, range 1.3 – 19.3%), while the average treatment HbF value was 23.9 ± 7.2 % (median 22.9%, range 10.2 – 40.7%). All 67 children increased their HbF in response to hydroxyurea therapy (median 16.7%, range 5.0 – 28.8%). There was a modest but statistically significant correlation between the baseline and treatment HbF (r=0.66, p<.0001). The estimated effect of one unit change in baseline HbF on treatment HbF was 1.11 (95% CI of 0.78, 1.43). When baseline %HbF was analyzed according to the beta globin haplotype, the overall ANOVA had a p-value of 0.02, indicating a statistically significant influence. Further analysis confirmed associations previously identified in adults with SCA, i.e. children with at least one copy of the CAR haplotype had a lower baseline HbF (5.9% vs 8.4%, p=.05), while those with at least one copy of the Senegal haplotype had a higher baseline HbF (11.1% vs 6.7%, p<.001). When hydroxyurea MTD (treatment) HbF values were analyzed according to beta globin haplotype while adjusting for baseline HbF, however, the effect of beta globin haplotype was not statistically significant (p=.13). Analyses of HbF according to alpha globin gene number revealed no statistically significant effects on either baseline or treatment HbF values. Taken together, these data support the hypothesis that beta globin haplotype significant influences baseline HbF values for children with SCA, but has no significant effects on hydroxyurea MTD HbF values. Accordingly, children with SCA should be offered hydroxyurea based solely on clinical indications, without consideration of baseline HbF or beta globin haplotype. Even children with low baseline HbF values or the CAR beta globin haplotype can respond to hydroxyurea therapy with an elevated %HbF. Future studies designed to identify genetic modifiers of treatment HbF values should focus on sequence polymorphisms in non-globin genes that have trans-acting effects on gamma globin gene expression.

scholarly journals Fetal hemoglobin in sickle cell anemia: relation to regulatory sequences cis to the beta-globin gene. Multicenter Study of Hydroxyurea

Blood ◽  
1996 ◽  
Vol 87 (4) ◽  
pp. 1604-1611 ◽  
Author(s):  
ZH Lu ◽  
MH Steinberg
Keyword(s):  
Gene Expression ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Regulatory Sequences ◽  
Beta Globin ◽  
Beta Globin Gene ◽  
Gamma Globin ◽  
Globin Gene Expression

Very different fetal hemoglobin levels among adult sickle cell anemia patients suggest genetic modulation of gamma-globin gene expression. In sickle cell anemia, different fetal hemoglobin levels are associated with distinct beta-globin gene haplotypes. Haplotype may be a marker for linked DNA that modulates gamma-globin gene expression. From 295 individuals with sickle cell anemia, we chose for detailed studies 53 patients who had the highest or the lowest fetal hemoglobin levels and 7 patients whose fetal hemoglobin levels were atypical of their haplotype. In these individuals, we examined portions of the beta- globin gene locus control region hypersensitive sites two and three, an (AT)x(T)y repeat 5′ to the beta-globin gene, a 4-bp deletion 5 to the A gamma T gene, promoters of both gamma-globin genes, 5′ flanking region of the G gamma-globin gene, and A gamma-globin gene IVS-II. Of the regions we studied all polymorphisms were always haplotype-linked and no additional mutations were present. This suggested that variations in these areas are uncommon mechanisms of fetal hemoglobin modulation in sickle cell anemia. Whereas unexamined cis-acting sequences may regulate gamma-globin gene transcription, trans-acting factors may play a more important role.

scholarly journals Sodium butyrate enhances fetal globin gene expression in erythroid progenitors of patients with Hb SS and beta thalassemia

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 454-459 ◽  
Author(s):  
SP Perrine ◽  
BA Miller ◽  
DV Faller ◽  
RA Cohen ◽  
EP Vichinsky ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Sodium Butyrate ◽  
Clinical Symptoms ◽  
Therapeutic Potential ◽  
Globin Gene ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Erythroid Progenitors ◽  
Gamma Globin

Increasing the expression of the gamma globin genes is considered a useful therapeutic approach to the beta globin diseases. Because butyrate and alpha-amino-n-butyric acid (ABA) augment gamma globin expression in normal neonatal and adult erythroid progenitors, we investigated the effects of sodium butyrate and ABA on erythroid progenitors of patients with beta thalassemia and sickle cell anemia who might benefit from such an effect. Both substances increased fetal hemoglobin (Hb F) expression in Bfu-e from 7% to 30% above levels found in control cultures from the same subjects with sickle cell anemia. The fraction of cultured erythroblasts producing Hb F increased more than 20% with sodium butyrate treatment in 70% of cultures. In most cultures, this produced greater than 20% total Hb F and greater than 70% F cells, levels which have been considered beneficial in ameliorating clinical symptoms. Alpha: non-alpha (alpha-non-alpha) imbalance was decreased by 36% in erythroid progenitors of patients with beta thalassemia cultured in the presence of butyrate compared with control cultures from the same subjects. These data suggest that sodium butyrate may have therapeutic potential for increasing gamma globin expression in the beta globin diseases.

scholarly journals Sodium butyrate enhances fetal globin gene expression in erythroid progenitors of patients with Hb SS and beta thalassemia

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 454-459 ◽  
Author(s):  
SP Perrine ◽  
BA Miller ◽  
DV Faller ◽  
RA Cohen ◽  
EP Vichinsky ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Sodium Butyrate ◽  
Clinical Symptoms ◽  
Therapeutic Potential ◽  
Globin Gene ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Erythroid Progenitors ◽  
Gamma Globin

Abstract Increasing the expression of the gamma globin genes is considered a useful therapeutic approach to the beta globin diseases. Because butyrate and alpha-amino-n-butyric acid (ABA) augment gamma globin expression in normal neonatal and adult erythroid progenitors, we investigated the effects of sodium butyrate and ABA on erythroid progenitors of patients with beta thalassemia and sickle cell anemia who might benefit from such an effect. Both substances increased fetal hemoglobin (Hb F) expression in Bfu-e from 7% to 30% above levels found in control cultures from the same subjects with sickle cell anemia. The fraction of cultured erythroblasts producing Hb F increased more than 20% with sodium butyrate treatment in 70% of cultures. In most cultures, this produced greater than 20% total Hb F and greater than 70% F cells, levels which have been considered beneficial in ameliorating clinical symptoms. Alpha: non-alpha (alpha-non-alpha) imbalance was decreased by 36% in erythroid progenitors of patients with beta thalassemia cultured in the presence of butyrate compared with control cultures from the same subjects. These data suggest that sodium butyrate may have therapeutic potential for increasing gamma globin expression in the beta globin diseases.

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