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.
Alpha (Α) thalassaemia is the most common inherited disorder in Malaysia. The clinical severity is dependant on the number of Α genes involved. Full blood count (FBC) and haemoglobin (Hb) analysis using either gel electrophoresis, high performance liquid chromatography (HPLC) or capillary zone electrophoresis (CE) are unable to detect definitively alpha thalassaemia carriers. Definitive diagnosis of Α-thalassaemias requires molecular analysis and methods of detecting both common deletional and non-deletional molecular abnormailities are easily performed in any laboratory involved in molecular diagnostics. We carried out a retrospective analysis of 1623 cases referred to our laboratory in Universiti Kebangsaan Malaysia Medical Centre (UKMMC) for the diagnosis of Α-thalassaemia during the period October 2001 to December 2012. We examined the frequency of different types of alpha gene abnormalities and their haematologic features. Molecular diagnosis was made using a combination of multiplex polymerase reaction (PCR) and real time PCR to detect deletional and non-deletional alpha genes relevant to southeast Asian population. Genetic analysis confirmed the diagnosis of Α-thalassaemias in 736 cases. Majority of the cases were Chinese (53.1%) followed by Malays (44.2%), and Indians (2.7%). The most common gene abnormality was ΑΑ/--(SEA) (64.0%) followed by ΑΑ/-Α(3.7) (19.8%), -Α(3.7) /--(SEA) (6.9%), ΑΑ/ΑΑCS (3.0%), --(SEA)/--(SEA) (1.2%), -Α(3.7)/-Α(3.7) (1.1%), ΑΑ/-Α(4.2) (0.7%), -Α(4.2)/--(SEA (0.7%), -Α(3.7)/-Α(4.2) (0.5%), ΑΑ(CS)/-- SEA) (0.4%), ΑΑ(CS)/ΑΑ(Cd59) (0.4%), ΑΑ(CS)/ΑΑ(CS) (0.4%), -Α(3.7)/ΑΑ(Cd59) (0.3%), ΑΑ/ΑΑ(Cd59) (0.1%), ΑΑ(Cd59)/ ΑΑ(IVS I-1) (0.1%), -Α(3.7)/ΑΑ(CS) (0.1%) and --(SEA) /ΑΑ(Cd59) (0.1%). This data indicates that the molecular abnormalities of Α-thalassaemia in the Malaysian population is heterogenous. Although Α-gene deletion is the most common cause, non-deletional Α-gene abnormalities are not uncommon and at least 3 different mutations exist. Establishment of rapid and easy molecular techniques is important for definitive diagnosis of alpha thalassaemia, an important prerequisite for genetic counselling to prevent its deleterious complications.
The capillary electrophoresis (CE) is a new system that utilizes the principle of electrokinetic separation of molecules in eight electrolyte buffer-filled silica capillaries. In this study, we established the normal ranges of haemoglobin A2 (HbA2) and haemoglobin F (HbF) levels for normal individuals using this system and also the HbA2 level in beta thalassaemia and haemoglobin E (HbE) individuals.