Sviluppo di una piattaforma per la diagnosi prenatale non invasiva di malattie genetiche in epoca gestazionale precoce

Prenatal diagnosis of aneuploidies and monogenic diseases is usually performed by amniocentesis or chorionic villous sampling. However, these procedures are associated with 0.5%-2% risk of miscarriage. The discovery of cell free fetal DNA (cffDNA) in maternal plasma in 1997 has provided a new source of fetal genetic material that can be safely obtained from maternal blood and successfully processed for non invasive genetic diagnosis (NIPD). In this study is described a new approach for non invasive prenatal diagnosis of β- thalassemia which is based on semiconductor sequencing (Ion Torrent PGM) and fetal haplotype inference. In particular, the approach is based on target sequencing of the mutation site, the β°39 non sense mutation of the HBB gene, and several informative SNPs spread in the β-globin gene cluster. The data analysis of each cffDNA sample and the inference of the most likely inherited haplotypes were determined by an automated pipeline which firstly constructs the parental haplotypes, using the sequencing data from the parental DNAs and an haplotype reference panel previously created. The pipeline, then, quantifies the allele counts observed in each site sequenced in the corresponding cffDNA sample and finally predicts the two haplotypes most likely inherited by the fetus using a hidden Markov Model (HMM). The results were finally compared with the sequencing data of the fetal DNA obtained by villocentesis. Using these approaches we have analyzed 30 out of 37 cffDNA samples; in seven samples, in fact, the pipeline could not proceed because of the lack of informative sites or of other parameters useful for downstream analysis. The fetal β°39 genotype was correctly predicted in 24/30 (80%) samples, while it was incorrectly defined in 6/30 (20%) cases. The incorrect results obtained in these last samples was due to the erroneus inference of the maternal haplotype. On the contrary, the paternal haplotype was correctly detected in all 30 samples processed. In the next future we are planning to improve the protocol by increasing the number of potentially informative SNPs and to process a higher number of Sardinian β°39 carriers in order to expand the haplotype reference panel. This haplotype-based approach has given encouraging results and we think that it could be a starting point for eventual future application of NIPD also to other monogenic disorders.