Detection of F8 Mutations by High Resolution Melting (HRM) Analysis.

Approximately 50% of severe and all moderate or mild haemophilia A (HA) mutations are single base substitutions, small insertions or deletions with over 900 different mutations having been described. The usual way to screen these HA cases is by DNA sequencing of all 26 exons which is time consuming and expensive.

We have developed the technique of HRM analysis using the Corbett Rotor Gene 6000 for successful high throughput screening of HA mutations. A target sequence was first amplified in the presence of a dsDNA intercalating fluorescent dye followed by in-tube melting analysis. Initially fluorescence is high, but diminishes as the temperature is raised and DNA dissociates into single strands. The observed ‘melting’ behaviour is characteristic of a particular DNA mutation.

In our study we looked at HA mutations in 23 exons,(exons 3–26). Genomic DNA samples from 100 HA patients and carriers were amplified using primers targeting the specific exons. We detected all of the mutations by HRM analysis and each had a distinct ‘melt’ curve which enabled the carriers to be successfully screened. We detected all the HAMSTerS known mutations and our 26 novel mutations in our cohort of patients.

Our results show that HRM could be used as the initial screening method followed by DNA sequencing of the amplicon where a different ‘melt’ curve was observed. It could also be used to rescreen ‘negative’ mutation patients very quickly. This technique is simpler, cheaper and quicker than DNA sequencing method. This method may therefore be particularly useful for prenatal diagnosis and carrier diagnosis.