FUNCTIONAL CHARACTERIZATION OF NOVEL DETERMINANTS OF HOLOPROSENCEPHALY (HPE)

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SUMMARY Congenital defects are a leading cause of morbidity worldwide, accounting for the deaths of 330,000 new- born every year. Brain malformations appear to be the most common congenital anomalies and are a major cause of death and lifelong disability. In the majority of cases the cause remains uncertain, due to the complexity and the multi-genic origin of these anomalies. Genes encoding Transcription Factors (TFs) and epigenetic regulators have become relevant candidates given the central role of these proteins in integrating signalling cascades and orchestrating multiple biological processes. Deficiency in their function may disturb entire transcriptional programs, involving several genes and molecular pathways. Here we combine mouse genetics and epigenomic approaches to uncover the role of PRDM15, a previously unsuspected disease-associated epigenetic regulator, in congenital brain malformations. Moreover, by functionally characterizing PRDM15 downstream effectors (e.g. NOTCH and WNT/PCP pathways) we uncover hitherto underappreciated genes mutated in patients with brain malformations (i.e. HPE and microcephaly). Preliminary data: We have characterized the function of PRDM15 in regulating the mouse naïve ESC state. We have then expanded our findings to demonstrate that, in vivo, PRDM15 depletion leads to: 1) embryonic lethality at E12.5-E14.5; 2) patterning defects affecting Anterior/Posterior patterning and forebrain development. In particular we have observed a failure to properly form the Axial Mesendoderm (AME), an embryonic structure necessary for proper anterior specification; 3) Finally, in collaboration with the groups of M.Muenke (NIH) and F.Hildebrandt (Harvard), we have identified heterozygous and homozygous mutations in PRDM15 linked to Holoprocensephaly (HPE) and microcephaly, respectively and mutations in over 100 PRDM15-regulated genes (~20% likely to be damaging) in a large cohort of HPE patients (132 trios and 188 singletons). In AIM1 we propose to define the molecular basis of PRDM15 function, specifically to understand how it regulates, at the level of chromatin, the transcriptional program driving forebrain development. Next, in AIM2 we will establish a causative link between human PRDM15 mutations, HPE-associated genetic variants identified as PRDM15-transcriptional targets, and the associated spectrum of brain malformations (ranging from HPE, to Microcephaly). The significance of these studies is that PRDM15 is a so far uncharacterized critical regulator of embryonic development and knowledge of the downstream regulated pathways will be useful to the field of regenerative medicine and will have diagnostic and clinical implications for patients with holoprosencephaly and microcephaly. The clinical Impact of these studies is that given the multigenic origin of HPE, targeted sequencing of PRDM15, and its key downstream targets, can potentially be added to routine genetic testing in families at risk of carrying other HPE-causing mutations (e.g. SHH, ZIC2, TGIF).