Sickle cell disease (SCD) is an inherited red cell disorder, characterized by the tendency of haemoglobin S or sickle haemoglobin to polymerize and assume a characteristic sickle shape. Molecular analysis has been the mainstay of detection method when confirmation is required. Previously a polymerase chain reaction (PCR)-based restriction enzyme analysis was used for this purpose. A simple bidirectional allele-specific amplification, recently described by Waterfall in 2001 was used to detect the GAG --> GTG mutation on codon 6 of the beta globin gene. Two sets of primers for the mutant and the wild type alleles were used in a single PCR reaction to amplify the regions of interest. The resultant PCR products will produce two fragments at 517 and 267 base pair (bp) respectively. This report highlights the investigations for SCD in the family of a 16-year old girl with recurrent painful crisis affecting the lower limbs whereby the family members are asymptomatic for the disease. Her haemoglobin electrophoresis at an alkaline pH showed dense bands at the HbS and HbF regions, while her father and two sisters had bands at HbS, HbF and HbA. The PCR analysis showed that she was homozygous for the mutation by the presence of only one band at 267 bp fragment, while the father and her sisters were heterozygotes, with the presence of two bands at 267 as well as 517 bp fragments. DNA sequencing of the sample confirmed the mutation. In conclusion, this case report highlighted the simple and cheap yet practical method for molecular confirmation of the presence of HbS gene in subjects with homozygous or heterozygous state of the condition.
Anemia, Sickle Cell/*diagnosis ; Anemia, Sickle Cell/*genetics ; Base Sequence ; Fathers ; Hemoglobin, Sickle/*genetics ; Heterozygote ; Homozygote ; Malaysia ; Mutation ; Nucleic Acid Amplification Techniques ; Pedigree ; Polymerase Chain Reaction ; Siblings

Medicine has, in recent years, incorporated wave after wave of new scientific discoveries and technologies. Molecular medicine is one of these technologies and shows a dramatic example of the impact of advances in basic science. Advances in molecular biology have revolutionized daily clinical practice, particularly in developed countries, such that practitioners who received their medical education decades ago now need to adapt to this new discipline. While molecular medicine may not be a priority health issue in developing countries such as Papua New Guinea, it is equally important to ensure that the basic principles of knowledge and understanding of what goes on in that field form part of today's teaching of all practitioners of medicine and allied health workers. The three papers in this series aim to present molecular biology and its medical applications in as simple and lucid a manner as possible so that its scientific basis and principles as well as its potential for diagnosis and management of diseases are well appreciated.
Allied Health Personnel - education ; Anemia, Sickle Cell - genetics ; Clinical Competence ; Cloning, Molecular ; DNA - genetics ; Papua New Guinea

Sickle cell disease (SCD) is a single gene genetic disease, which seriously threatens the life span and quality of patients. On the basis of the pathogenesis of SCD and the alternative therapy based on fetal hemoglobin F (HbF), the research progress of transcription factors involved in the regulation of HbF gene expression, such as BCL11A, ZBTB7A, KLF-1, c-MYB and SOX6, as well as the application of CRISPR / Cas9, TALEN, zinc finger nuclease and other gene editing technologies in this field has been made, providing a solid theoretical basis for the exploration of new treatment schemes for β- like hemoglobin diseases, such as sickle cell disease and β- thalassemia.
Anemia, Sickle Cell/therapy* ; Cell Line, Tumor ; DNA-Binding Proteins ; Fetal Hemoglobin/genetics* ; Genetic Therapy ; Humans ; Repressor Proteins/genetics* ; Transcription Factors

OBJECTIVETo investigate the effects of combined use of low-dose hydroxyurea (HU) and sodium butyrate (NaB) on the expression of 7 globin genes (zeta, alpha, epsilon, Ggamma, Agamma, delta, and beta) in human erythroid progenitor cells.

METHODSHuman erythroid progenitor cells were cultured using a two-step liquid culture system and treated with HU and NaB either alone or in combination. The inhibitory effects of the agents on the cell growth were monitored with trypan blue exclusion assay, and the changes in the mRNA of the 7 globin genes were detected using RT-PCR.

RESULTSLow-dose HU combined with NaB resulted in significantly lower inhibition rate of the erythroid progenitor cells than routine dose HU and NaB used alone (28.56% and 38.80%, respectively, P<0.05). Compared with untreated cells (0.653-/+0.092 and 0.515-/+0.048), HU combined with NaB significantly increased the expression of Ggamma-and Agamma- mRNA (1.203-/+0.018 and 0.915-/+0.088, respectively, P<0.05), and HU and NaB used alone produced similar effects (1.305-/+0.016 and 0.956-/+0.029 for HU, and 1.193-/+0.070 and 0.883-/+0.012 for NaB, P>0.05). HU and NaB, either used alone or in combination or at different doses, caused no significant changes in the other globin genes (zeta, alpha, epsilon, delta and beta) (P>0.05).

CONCLUSIONLow-dose HU combined with NaB can up-regulate gamma globin gene expression, especially Ggamma-mRNA expression, to decrease the growth inhibition on human erythroid progenitor cells in vitro, but produces no significant effect on the expressions of zeta, alpha, epsilon, delta and beta genes.

Anemia, Sickle Cell ; genetics ; Butyrates ; administration & dosage ; pharmacology ; therapeutic use ; Cells, Cultured ; Drug Therapy, Combination ; Erythroid Precursor Cells ; cytology ; drug effects ; physiology ; Erythropoiesis ; drug effects ; Humans ; Hydroxyurea ; administration & dosage ; pharmacology ; therapeutic use ; RNA, Messenger ; genetics ; metabolism ; gamma-Globins ; genetics ; metabolism