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Responsabile: dott.ssa Anna Savoia
Background
Inherited disorders in which the production of all (pancitopenia) or even one (anemia, neutropenia, and thrombocytopenia) blood cell types ceases or is greatly reduced are heterogeneous diseases characterized by complex phenotypic aspects. Clinical features often overlap, making diagnosis difficult to attain. For these reasons, they are undoubtedly underdiagnosed in both pediatric and adult hematology practices, which has often led to treat patients with unnecessary or even dangerous therapies. Sequential, multi-step approaches have been developed and validated to specifically recognize the different diseases but despite many efforts, a relatively high frequency of patients does not receive a definitive diagnosis for lack of suitable samples or complexity of procedures. Even when clinical and laboratory approaches lead to a diagnosis, identification of the diseasecausing mutations remains an arduous task because of genetic heterogeneity and/or genes with multiple exons, harboring a wide spectrum of mutations spread throughout the gene. To bypass complex algorithms, new sequencing technologies, such as next-generation sequencing (NGS) platforms, are very promising because they could meet the demand of rapid and accurate diagnostic processes, as required in this and other field of medicine.
Aims
NGS is a massive parallel sequencing technology that allows sequencing of thousands of large regions of the genome rapidly. As costs and efforts of these new technologies are still expensive, we will focus on the smallest design that allows the sequencing of approximately two hundred genes. Taking advantage of our large series of patients with bone marrow failure syndromes (BMFS) or thrombocytopenias collected in many years of experience in this field, we propose to develop and validate a NGS assay to sequence all genes known to be responsible for these diseases.
Study Design
The Illumina sequencing technology will be used for the development of a NGS assay in collaboration with Dr Germana Meroni at CBM (Cluster in Biomedicine, Trieste), where a platform is available. In the first year, we will generate a first set of oligonucleotides for targeting the regions of interest, which include coding and non-coding exons of the genes involved. After DNA library preparation of 96 selected individuals, including patients with known mutations, amplification will allow cluster generation for sequencing. Database will be generated for analyses, which will include coverage index of the targets, filtering of variants from SNP databases, and validation of potential variations.
Regions of interested are usually variably covered depending on different variables, primarily the probes used. To increase efficiency, a new set of oligonucleotides will be generated. In case of a further partial coverage, the third year of will be dedicated to both improve efficiency and sequence uncharacterized patients from our database.
Summary Preliminary Data
Thanks to the collaborations with Drs Carlo Balduini (IRCCS San Matteo, Pavia) and Carlo Dufour (IRCCS Gaslini, Genova), we banked DNA samples of more than 600 unrelated probands affected with inherited BMFS or thrombocytopenias, such as Fanconi anemia, Diamond-Blackfan anemia, amegakaryocytic thrombocytopenia, MYH9-related disease, Bernard-Soulier syndrome. Whereas in more that 200 cases, molecular genetic testing identified the mutations and the genes involved, in the other the diagnosis remains unknown despite the application of the diagnostic algorithms.
Materials and methods
We will apply protocols from Illumina to enrich regions of the genes. The enriched DNA will be sequenced using the HiScanSQ system, which integrates the power and resolution of the NGS with the high-throughput capacity. The sequence analysis will consist of three phases, (1) aligning and recalibrating reads; (2) calling sequence variants using various algorithms; (3) filtering set of variants. Once known mutations of the controls are confirmed, potential alterations in the other undiagnosed samples will be validated using PCR and Sanger sequencing.
Main Expected Results and Impact
The following results are expected from this project:
1) Development of the NGS will uncover the genes and the pathologic mutations directly from the DNA of an affected individual
without the need of any other investigations.
2) Whenever no mutation is identified, patients and their families will be regarded as affected with new forms of BMFS or thrombocytopenias.
They will represent a selected group more suitable for cloning of novel genes.
3) Once validated, the NGS assay could have a commercial value and be used as a diagnostic kit.
Significance and Relevance for National Health System (SSN): The National Health System will benefit from the use of a NGS approach because it will enable a quick and efficient molecular diagnosis, directly from DNA samples, which can be easily shipped to national centralized laboratories, with no prior requirement for carrying out any investigations. Even if designed for relatively rare diseases, this tool would have a more extended range of applications to acquired forms, which are more frequent and diagnosed after exclusion of the inherited ones.