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Jeudi 9 Décembre, Juliette Albuisson (Service de Génétique Médicale CHU Nantes) Print
Written by Pierre PETERLONGO   

Unscrambling regulation of human F8 gene by NGS and non coding mutation analysis in haemophilic patients

10h30 Salle Aurigny

In human genetics, the recent development of Next-Generation Sequencing (NGS) technologies allows the screening of all coding sequences of a genome (ie: an exome) with litte time and wet lab use, in the perspective of disease-related gene discovery. NGS can also provide several megabases of a targeted genomic region surrounding a gene, which can lead to the discovery of cryptic, previously undetectable mutations in various hereditary disorders. Haemophilia A (HA), a frequent X-linked hereditary disorder, is due to coagulation factor VIII (F8) deficiency. Using 2nd generation sequencing technologies (Sanger Sequencing) and several other low- or medium-throughput techniques, we can identify F8 gene sequence alteration in 95-98% of families, including 2 recurrent inversions disrupting intron 1 or intron 22 of the gene (accounting for 55% of severe cases), coding or splice-site point mutations, and various deletions/insertions. In a small number of cases, however, we and others are unable to find any F8 coding or disrupting mutation. This result points to possible undetected rearrangements or regulatory point mutations around F8 that alter its proper expression.

We have selected 10 true recessive X-linked HA families with no known mutation. We will (1) sequence a region spanning ~5 Megabases including F8, in order to get the complete region genotype including the expected deleterious sequence variation(s), in one male patient from each family. We will (2) exclude all known polymorphic variations by filtering through polymorphisms databases (dbSNP, DGV…). Variants will then be selected (3) based on their functional effects prediction, by combining various bioinformatic analyses: phylogenic sequence conservation and complexity, transcription factors binding sites (motif search and discovery) ad their tissue distribution, etc… The selected variants will (4) be tested in a cellular system expressing F8 gene, to confirm their predicted deleterious effect. This analysis will shed light on the regulatory system of F8 gene which is still widely unknown.

The in silico study design is of great importance in our analysis, from the region boundaries setting to the variants selection, through the sequence enrichment process. Achievement of such a project needs a close collaborative work between bioinformatics and genetics experts.

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