Nuovi approcci molecolari per lo studio di malattie monogeniche rare: utilizzo dell’exome sequencing per la ricerca di geni malattia

As defined by European Union, rare diseases are a broad range of disorders with prevalence ≤ 5/10000 in general population. Some rare diseases are the result of bacterial or viral infections, allergies and environmental causes, or are degenerative and proliferative, but the most have a genetic etiology. Discovering alleles underlying genetic conditions is essential for the comprehension of the pathogenesis and for the prevention of the disease through identification of carriers and prenatal diagnosis. Conventional strategies for disease gene discovery, like positional cloning and Sanger sequencing of candidate genes, have led, to date, to detect the genetic detrminants of about 3000 Mendelian phenotypes, representing about the 50% of genetic phenotypes known. In recent years many genes causing human genetic disorders have been identified through the analysis of the variants revealed by whole-genome or exome sequencing by “Next-Generation” sequencing technologies (NGS). Herein, we report the results of an exome sequencing project applied to the identification of the gene responsible for a rare and genetically heterogenous ciliopathy, the Jeune Syndrome (Asphyxiating thoracic dystrophy, JATD, MIM 208500). Exome sequencing has been performed on the affected and respective parents of two unrelated Sardinian families in which genetic basis of the disease was not yet elucidated. Exome variants analysis strategies led us to identify a missense substitution in DYNC2H1 (MIM 603297), a gene involved in intraflagellar transport pathways of cilia that has been previously associated to the disease (Dagoneau et al., 2009; Merrill et al., 2009). The substiution has been found in homozygosity in the probands of one of the two families JATD and in heterozygosity in their parents. To date, exome analysis is still in progress in the second family JATD. For this family, there was only one gene that has been considered a good candidate for the disease, MAP1S (MIM 607573), that codifies for a microtubule associated protein. Unfortunately, the compound heterozygous state for two variants, revealed by exome sequencing in the proband, was not validated by variants resequencing with Sanger method. No deleterious variants have been found in DYNC2H1 but the involvement of this gene in disease pathogenesis cannot be excluded because of the failed capture of some exons, that will be resequenced by NGS technologies. Studies in vivo and in vitro could explain the functional impact of the DYNC2H1 missense substitution identified by exome sequencing on the protein function.