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High throughput molecular diagnostics in individual patients for genetic diseases with heterogeneous clinical presentation

Periodic Report Summary - TECHGENE (High- throughput molecular diagnostics in individual patients for genetic diseases with heterogeneous clinical presentation)

Publishable summary:

The European project TECHGENE aims to develop and implement High-throughput sequencing (HTS) techniques in clinical genetic testing. In this process, ethical and economic aspects are also addressed, ultimately leading to uniform guidelines for diagnostic labs on how to implement and use HTS techniques in genetic testing.

A stepwise approach is used for the model diseases studied:
first-level mutation analysis of patient samples by classic methods, to identify samples to be used in subsequent steps
development and application of HTS reagents and protocols on patient samples with known and unknown mutations
validation of protocols
development of read-out and data-analysis tools

During the first 18 months the focus has been on the first 2 steps. For hemoglobinopathy and breast cancer (WP1), HTS techniques have been developed and tested for the genes involved; Hemoglobinopathy patient samples were screened. For breast cancer genes, two different mutation scanning methods were evaluated and enrichment steps developed and tested.

For the sensory disorders (hearing loss, retinal dystrophies and Usher syndrome, WP2) research activities focused on the development of HTS approaches: For hearing loss patients, an initial prototype non-Next generation sequencing (NGS)-based mutation screening technique was designed. However, in the course of the first reporting period the strategy has been altered to use NGS-based techniques. For retinal dystrophies, a first level screening of patients, using appropriate APEX arrays and further development of APEX arrays, was performed. The development of a medical re-sequencing protocol on the genes responsible for Autosomal recessive retinitis pigmentosa (ARRP) and LCA is now in progress. For the Usher syndrome a NGS-based approach has been designed, developed and tested using different platforms.

For paraplegia and ataxia (WP3), HTS approaches including direct array-based re-sequencing (paraplegias) and NGS-based re-sequencing using gene capture by arrays (ataxia) have been developed. A joined gene capture tool for genetic movement disorders is now being developed for recessive ataxia, paraplegia, and will be extended to dystonia and hereditary Parkinson. In total, over 100 genes will be included in the design. Also, several alternative gene capture enrichment strategies have been tested and employed, including flow through arrays, and Raindance.

As a first level Copy number variant (CNV) analysis, Multiplex ligation-dependent probe amplification (MLPA) probe sets have been developed for many Mental retardation (MR) associated CNVs and tested on 1000 patients with unexplained mental retardation. For MR (WP4), HTS technology is developed and the performance compared with microarray-based techniques. Exome sequencing was explored and turned out to be an excellent tool to identify mutations in rare MR syndromes. In addition, different enrichment strategies have been compared.

Considerable progress has been made in optimising HTS technology for MR. However, in comparison to genomic microarrays HTS technology does not yet have the robustness and throughput for the detection of copy number variation as microarrays do. Microarrays will therefore remain the golden standard for the detection of this type of genomic variation until HTS technology improves considerably. Additional work has been focused on development of a data analysis tool. In-house data-analysis pipelines for NGS have been established and obtaining, storing and interpretation of next generation sequencing data are feasible.

A template for the ethical follow up of all ethical aspects of the project and a general information for the project website on ethical aspects have been made. As there are only few ethical and legal requirements for HTS for different countries, a specific analysis has been started and will be completed in collaboration with the public and professional policy committee of the European society of human genetics (WP5).

To evaluate the cost effectiveness and economic impact of introducing a new HTS diagnostic technology into clinical practice (WP6), a work plan for the second and third year has been prepared. Breast cancer diagnostics was selected for the first model and methods used for breast cancer diagnostics have been studied. Data to populate the economic model have been gathered.

The TECHGENE consortium organised several training and dissemination activities (WP7); 4 workshops, for TECHGENE people and external people (topics were next generation sequencing data analysis and enrichment strategies and breast cancer diagnostics). A training facility was set up in Prague and several research fellows have been trained onsite. Knowledge was disseminated in various ways, such as via the TECHGENE website ( scientific symposia, publications, etc. Furthermore, a NGS diagnostics knowledge network is being set up.