Use of next generation sequencing for isolated and syndromic Anophthalmia, Microphthalmia and Coloboma (MAC): a new approach to molecular genetic diagnosis

ABSTRACT: Use of Next Generation Sequencing for isolated and syndromic Anophthalmia, Microphthalmia and Coloboma (MAC): a new approach to molecular genetic diagnosis Department in Cellular Biotechnologies and Hematology PhD in Human Biology and Medical Genetics Supervisor: Prof. Pizzuti Antonio Correlator: Prof. Novelli Antonio Phd student: Dott. Iapichino Giuseppe In these last few years, both mendelian and complex diseases have reached a higher level of accuracy in the analytical process, with a more efficient diagnostic definition and also a more detailed genotype-phenotype correlation. Complex syndromic pathologies, such as microphthalmia and anophthalmia, often accompanied by complex clinical pictures, and characterized by a high level of genetic and phenotypic heterogeneity, can now be handled in a faster and thorough manner. Currently, national or international shared guidelines have yet to be defined and a phenotypic or genetic classification of these diseases is still missing. Similarly to what happens for most rare diseases, the diagnostic and care management of children with ocular syndromes suffers heavily from the absence of strong scientific knowledge that would help in the development of shared guidelines, mainly because the phenotypic heterogeneity of these diseases is massive. The aim of this project was to carefully investigate the genes involved in microphthalmia, anophthalmia, coloboma and related syndromic diseases using Next Generation Sequencing. This new molecular approach allows to make a massive sequencing of different genomic regions. The possibility to analyze millions of sequencing reactions in parallel at very reduced costs, with shorter processing times and very little amounts of initial DNA, has provided a formidable boost for the study of rare diseases. In the present study, a panel of different genes involved in the ocular development has been designed to simultaneously and rapidly analyze multiple ocular genes of a patient by using the Illumina-Nextseq 500 platform. NGS allowed us to analyze several genes for a given patient and to identify variants both in genes directly involved in the pathogenesis of anophthalmia, microphthalmia and coloboma, both in genes not directly implicated in syndromic or isolated MACs. This allowed us to understand at a molecular level many of those phenotypes described as shaded, atypical or not perfectly defined in a specific syndrome. In this regard, the continuous interaction with the referring physicians has been fundamental in our study, highlighting the importance of the collaboration between the lab scientist and the clinical geneticist that will eventually help to find a link between the phenotypes described and the many variants still to be identified.