DNA diagnostics for deafness using next generation DNA sequencing.

Although over the last 10 years the identification of genes for monogenic deafness has been very successful, diagnostic applications are lagging behind. The main obstacle for better DNA diagnostics is the high cost of DNA sequencing. In the current project we will develop a novel molecular diagnostics approach targeted to hereditary deafness, with the goal of providing high throughput, sensitive, reliable and cost effective diagnostic tests. We aim to develop reliable DNA diagnostics comprising all 30 known genes for autosomal recessive deafness on a 454 GS-FLX DNA sequencer (Roche). The method will enable usto analyse these deafness genes within a few weeks at an affordable cost. Therefore, a genetic diagnosis should be obtained in a much higher percentage of patients compared to current methods. Multiplex PCR will be performed for all exons of the genes. Specific sets of primers are designed carefully to pool amplicons in the same PCR reaction. Reaction conditions for each of these multiplex PCR reactions will be optimized. After validation, up to sixty different tags are available to distinguish patient's DNA, so batches of up to 60 patients will be sequenced in a single run. After the method has been validated, we willanalyse 350 patients using this system. All patients included will have non-syndromic, moderate to profound HL, and unaffected parents from European ethnicity. The results of this analysis will teach us the relative contribution of the different genes to ARNSHL in Europe, data which are currently unavailable, and which are very important for DNA diagnostics.

Keywords:DEAFNESS, DNA DIAGNOSIS

Disciplines:Genetics, Systems biology, Molecular and cell biology