Diagnosis of Sickle Cell Disease by Molecular Biology &Convential Technique among Sudanese Population

Abstract

Sickle cell disease is a major health problem worldwide particularly in developing countries including Sudan. The disease has a great impact on both individual and society. Sickle cell disease disorder produces prominent clinical manifestations. Subjects with heterozygous form (AS) are designated sickle cell trait; they are essentially healthy unless exposed to extreme conditions. Further more AS subjects are known for their immune resistance to justify malaria Patients of sickle cell anemia are known not to survive beyond their 20th birthday. Sickle-cell hemoglobin (HbS) is found as an inherited abnormality with relatively high frequency in many races and tribes, particularly that resident in, or originating from, the malarial regions of the world. Molecular diagnosis of the disease, genetic and family studies of patients becomes an important tool for management of sickle cell disease patients. In this study we compare the efficiency of conventional cellulose acetate electrophoresis techniques with recently developed molecular biology methods i.e. (RFLP, ARMS/PCR, RAPD).We also estimated the level of SCD amongst Sudanese populations living in central Sudan. The objectives of the study also is to provide a database for the establishment of the most appropriate genetic counseling services for SCD patients and their families and to identify areas of research and collaboration.The result of this study shows higher frequencies of sickle cell disease in certain Sudanese tribes. The level of sickle cell disease was found with higher frequencies in the Afro/Arabs groups living in western part of the country. The hemoglobin levels of the Sudanese sickle cell patient was found significantly lower than normal percentages in individuals with normal hemoglobin, the significant elevated hemoglobin F level was also observed among sickle cell patients which help making the disease less severe and contribute to the mildness of the SCD among Sudanese patients. The sickle cell gene was found with higher frequencies in certain Sudanese tribes. In the results of ARMS PCR, we have two lines M for mutation bands and N for normal bands. If we have two bands in M&N sample is heterozygous but if we have one band in M that means it is a homozygous for Hb S and if we have one in N line that means it is normal. Discrimination of HbAA, HbAS and HbSS using ARMS-PCR shows amplification of HbA and HbS genotypes using ARMSPCR, typing of the AA, AS, SS genotypes yielded the 207bp. The results of RFLP In this method after the amplification of DNA with primers1 (mutant), primer 2 (normal primer), the 281bp fragment was achieved, after that we digested this fragment with DdeI restriction enzyme in two fragment (200bp&81 bp) so +/+ shows that our sample is normal and we had 200 & 81 bp both of them but we couldn't see 80 bp so we just had 200 bp! For heterozygous carrier we had 3 bands 200&281&81 that we saw just 2 bands (200 and 281), that was -/+! for homozygous disease we had just one band 281 bp that shows our restriction sits disturb and our enzyme couldn't digest it