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