Development and implementation of a real-time reverse transcriptase polymerase chain reaction assay to detect the intron 22 inversion in haemophilia A patients

[摘要] English: Background: Haemophilia A is a bleeding disorder with an incidence rate of one in 5,000-10,000 males worldwide. The disorder has a substantial impact on patients and their caregivers and is underdiagnosed in developing countries such as South Africa, where there is a lack of genetic research in the field. Haemophilia A is caused by mutations in the FVIII gene, with the most common mutation in severe haemophilia A patients being inv22 (45%). The current inv22 detection methods have disadvantages. The aim of this study was to develop novel inv22 detection methods that could overcome the disadvantages of the current inv22 detection methods.Methodology: We recruited three controls, including one non-haemophilic volunteer (C1) and two non-related inv22 confirmed severe haemophilia A patients (C2 and C3). The controls were used to evaluate the inv22 I-PCR detection method and to develop a conventional and a real-time RT-PCR inv22 detection method, respectively. The controls were Sanger sequenced to confirm the results of the PCR detection methods. A further 60 participants (35 haemophilia A patients, 18 possible haemophilia A carriers and seven healthy volunteers) from central South Africa were recruited for this study. The newly optimised detection methods were used to screen the 60 participants for the inv22. The PCR results for the 60 participants were confirmed with Sanger sequence analysis. The inv22 results for the conventional RT-PCR, real-time RT-PCR and Sanger sequence analysis in this study were subsequently compared.Results and discussion: The inv22 I-PCR detection method could not be evaluated after several attempts, and after troubleshooting measures were exhausted. The main reason for this failure was contributed to the fact that the inv22 I-PCR detection method did not have any 'built-in' quality control steps that allowed for successful troubleshooting. The need for an uncomplicated inv22 detection method was thus, further motivated. The inv22 conventional and real-time RT-PCR detection methods were successfully optimised. Control C1 was confirmed to be inv22 negative and controls C2 and C3 to be inv22 positive with Sanger sequence analysis. The novel detection methods overcame some of the disadvantages associated with the previous detection methods, especially referring to cost and turnaround time. The two newly developed methods were used to screen the 60 study participants for the inv22. The results for the 60 study participants were confirmed with Sanger sequence analysis. When the different detection methods' results were compared, only a single difference was found between the two assays. The inv22 was found in 29.41% of our severe haemophilia A population.Conclusion: Rapid, accurate, reliable and cost-effective inv22 conventional RT-PCR and real-time RT-PCR detection methods were developed and validated. The newly developed inv22 detection methods overcome the disadvantages of the current inv22 detection methods and will allow for the wide-spread screening of haemophilia A patients and possible carriers for the inv22. In this study, the inv22 was found in only 29.41% of severe haemophilia A patients in our population, which was considerably lower than the globally reported 45%, however, this may be attributed to the relatively small sample size of the study.

[发布机构] University of the Free State