Molecular characterisation of Haemoglobin Constant Spring and Haemoglobin Quong Sze with a Combine-Amplification Refractory Mutation System

@#Background: The interaction of the non-deletional α+-thalassaemia mutations Haemoglobin Constant Spring and Haemoglobin Quong Sze with the Southeast Asian double α-globin gene deletion results in non-deletional Haemoglobin H disease. Accurate detection of non-deletional Haemoglobin H disease, which is associated with severe phenotypes, is necessary as these mutations have been confirmed in the Malaysian population. Methods: DNA from two families with Haemoglobin H disease was extracted from EDTAanticoagulated whole blood and subjected to molecular analysis for α-thalassaemia. A duplex polymerase chain reaction was used to detect the Southeast Asian α-globin gene deletion. Polymerase chain reaction-restriction fragment length polymorphism analysis was then carried out to determine the presence of Haemoglobin Constant Spring and Haemoglobin Quong Sze. A combine- amplification refractory mutation system protocol was optimised and implemented for the rapid and specific molecular characterisation of Haemoglobin Constant Spring and Haemoglobin Quong Sze in a single polymerase chain reaction. Results and Conclusions: The combine- amplification refractory mutation system for Haemoglobin Constant Spring and Haemoglobin Quong Sze, together with the duplex polymerase chain reaction, provides accurate pre- and postnatal diagnosis of non-deletional Haemoglobin H disease and allows detailed genotype analyses using minimal quantities of DNA.

The use of the amplification refractory mutation system (arms) in the detection of rare beta-thalassemia mutations in the Malays and Chinese in Malaysia.

Molecular characterization and prenatal diagnosis for beta-thalassemia can be carried out using the Amplification Refractory Mutation System (ARMS). The ARMS is a rapid and direct molecular technique in which beta-thalassemia mutations are visualized immediately after DNA amplification by gel electrophoresis. In the University of Malaya Medical Center, molecular characterization and prenatal diagnosis for beta-thalassemia is carried out using ARMS for about 96% of the Chinese and 84.6% of the Malay patients. The remaining 4% and 15.4% of the uncharacterized mutations in the Chinese and Malay patients respectively are detected using DNA sequencing. DNA sequencing is an accurate technique but it is more time-consuming and expensive compared with the ARMS. The ARMS for the rare Chinese beta-mutations at position -29 (A-->G) and the ATG-->AGG base substitution at the initiator codon for translation in the beta-gene was developed. In the Malays, ARMS was optimized for the beta-mutations at codon 8/9 (+G), Cap (+1) (A-->C) and the AATAAA-->AATAGA base substitution in the polyadenylation region of the beta-gene. The ARMS protocols were developed by optimization of the parameters for DNA amplification to ensure sensitivity, specificity and reproducibility. ARMS primers (sequences and concentration), magnesium chloride concentration, Taq DNA polymerase and PCR cycling parameters were optimized for the specific amplification of each rare beta-thalassemia mutation. The newly-developed ARMS for the 5 rare beta-thalassemia mutations in the Chinese and Malays in Malaysia will allow for more rapid and cost-effective molecular characterization and prenatal diagnosis for beta-thalassemia in Malaysia.

Significance of lateralized focus of intra-carotid arterial diazepam injection for intractable epilepsy with bilateral synchronous epileptiform discharges / 第三军医大学学报

Objective To explore the significance of intra carotid arterial diazepam injection in lateralization of intractable epilepsy with bilateral synchronous epileptiform discharges. Methods Intra carotid arterial diazepam injection test was performed in 20 patients suffering from intractable epilepsy with bilateral synchronous epileptiform discharges under EEG monitoring. Side of the epileptogenic focus was determined by observation of the inhibition of epileptiform discharges after injection. Results After diazepam injection, disappearance of bilateral synchronous epileptiform discharges was found in 16 patients (80%), so the injection side was decided as the epilepogenic focus. Disappearance of the epileptiform discharges was found at the injection side, but disappearance of the bilateral epileptiform discharges was found after injection at the opposite side in 4 cases (20%). The accuracy of lateralization was proved by surgery, electrocorticography (EcoG), and depth electroencephalography (DEEG) in 12 out of the 20 cases. During the follow up for 1-6 5 years after surgery and gamma knife treatment, disappearance of clinical seizure and epileptiform discharges of EEG and improvement were found in 15 cases (75%) and 5 cases (25%), respectively. Conclusion Intra carotid arterial diazepam injection is of important clinical value in lateralization of the epileptogenic focus before surgery for patients with intractable epilepsy.