Phd in HUMAN BIOLOGY AND MEDICAL GENETICS

Thesis title: GENETIC AND MOLECULAR INVESTIGATIONS ABOUT THE AETIOPATHOGENESIS OF MAYER-ROKITANSKY-KÜSTER-HAUSER SYNDROME: A ROLE FOR PROTEIN KINASE X

Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is a rare disease, characterised by the agenesis of vagina and uterus in women with a normal 46,XX karyotype. Most cases are sporadic, but familial recurrence has also been described. Currently, genetic alterations associated to MRKH syndrome have been detected only in a small percentage of patients. A cohort of Italian unrelated MRKH patients was investigated to explore the presence of pathogenic copy number variations (CNVs) by array-CGH and MLPA analysis. On the whole, aberrations were found in 9/36 (25%) patients. Microdeletions previously associated to MRKH syndrome, at 16p11.2 (encompassing TBX6 gene) and 17q12 (including LHX1 and HNF1B genes) chromosomal regions, were detected in two cases; a third patient showed a duplication of CNE-2, a SHOX enhancer. Another patient carried a novel heterozygous microduplication in Xp22.33, not yet described in MRKH patients, containing the PRKX gene, which encodes for a serine/threonine cAMP-dependent protein kinase. To predict the potential significance of CNVs in MRKH pathogenesis, we provided a network analysis for protein-coding genes found in the altered genomic regions. Although not all of these genes taken individually showed a clear clinical significance, their combination in a computational network highlighted that the most relevant biological connections are related to anatomical structure development. Moreover, quantitative RT-PCR experiments were conducted on RNA samples from vaginal vestibule tissue of MRKH patients, demonstrating altered expression of PRKX and other selected candidate genes. Using in vitro modelling, we proved that ectopic PRKX overexpression promotes cell motility through epithelial-to-mesenchymal transition (EMT) activation in a vaginal keratinocytes cell line, fundamental processes in urogenital tract morphogenesis. Moreover, our findings showed that PRKX overexpression is capable of deregulating transcriptional levels of HOX genes implicated in urinary and genital tracts development. In conclusion, the results described in the present study identified novel genetic alterations and interactions that may be likely involved in MRKH phenotype determination, so adding new insights into the complex puzzle of MRKH disease.