Gene-environment interactions in common craniofacial malformations

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Everson, Joshua Lucas, author

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Online

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Summary

Unraveling the complex interactions between genetic and environmental influences that cause birth defects is necessary for identifying sensitive populations and developing prevention strategies. Th...

Unraveling the complex interactions between genetic and environmental influences that cause birth defects is necessary for identifying sensitive populations and developing prevention strategies. This dissertation focuses on two etiologically-complex and debilitating human craniofacial birth defects, orofacial clefts (OFCs) of the lip and palate and holoprosencephaly (HPE). The objective of this body of work was to identify specific etiologically-relevant genetic and environmental factors and to investigate their interaction in the genesis of these birth defects. These studies focused upon Sonic Hedgehog (Shh) signaling because this pathway is a critical regulator of craniofacial morphogenesis and is sensitive to both genetic and environmental disruption. First, complementary in vivo and in vitro models of Shh pathway modulation were utilized to identify Shh pathway targets in the cranial neural crest-mesenchyme of the facial growth centers that form the upper lip. Comparative transcriptomic analysis identified biological processes regulated by Shh signaling during facial morphogenesis, including previously unrecognized roles in perivascular biology and angiogenesis. This approach also identified several Shh-regulated genes that serve as candidate human orofacial clefting genes. Illustrating the utility of this approach, we demonstrated that Shh directly regulates Foxf2 which drives cranial neural crest cell proliferation during lip morphogenesis and cleft pathogenesis, and that polymorphisms in FOXF2 are associated with cleft lip in humans. We then investigated specific gene-environment interactions in the genesis of HPE, a malformation of the forebrain that commonly co-occurs with OFCs. We demonstrated for the first time that homozygous mutations in the Shh pathway transcriptional activator Gli2 cause HPE in the mouse, and that normally-silent, single-allele Gli2 mutations lower the threshold for teratogen-induced HPE. These findings set the stage for investigating the teratogenic potential of environmentally-relevant Shh pathway inhibitors. We focused upon the pesticide synergist piperonyl butoxide (PBO) because this compound was reported to inhibit the Shh pathway, has widespread and increasing commercial and household use, and had not been rigorously examined for potential developmental toxicity. We confirmed that PBO is a potent inhibitor of the Shh pathway and that a single dose targeted to forebrain and face development causes full blown HPE in the mouse. Highlighting potential gene-environment interactions, we also demonstrated that the teratogenic effect of PBO is exacerbated in mice with normally-silent, single allele mutations in Shh. Dose-response assays demonstrated a lowest observable effect level for PBO-induced malformations more than 30-fold lower than the value currently utilized in risk assessment. These findings illuminate the etiological complexity of OFCs and HPE and provide a framework to understand how variable outcomes--from apparently normal to severely affected-- are often observed in a single human pedigree. By providing specific genetic and environmental risk factors, these studies also inform prevention strategies based upon identification of sensitive populations and specific interacting environmental factors.

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