DISCUSSION
Precision medicine utilizing genomic methods implies that medical geneticists can effectively navigate a series complex interactions that ultimately induce malformation conditions such as HPE. The challenges to this effort are many. These can include, in part, the variable quality of the data, the impulse to over-filter datasets in an effort to simplify the genetic interpretation, the lack of clear criteria for driver mutations as either sole or multifactorial factors (e.g. digenic inheritance), the paucity of models for determining modifier effects, the frequent lack of clinical details of the family structure and affection status, any teratogen/environmental exposure considerations, the incomplete understanding of potential gene:gene and/or gene:environment interactions, the lack of effective guidelines for non-coding variation, the tendency to ignore the key role of the timing of these interacting factors, as well as the continuously evolving considerations attempting to foster an integrated interpretation into medical practice. Extrapolating from lessons gleaned from the example of carcinogenesis clearly supports the role of considering both genes and the environment. This is almost certainly the case with the pathogenesis of HPE. Furthermore, the genetic variation of human SMO is now clearly implicated in both rare developmental malformations, as well as somatic variants in human cancer. We now argue that the knowledge of the Hh pathway components with respect to their fundamental sequence conservation and their gene regulatory relationships provides insight into both.
Herein, we investigated the role of the SMO gene in craniofacial and forebrain development using zebrafish as a model system. This study is intended as a comprehensive analysis of variants detected in a malformation (HPE) known to be caused by disturbed function of the hedgehog pathway. In doing so, our study directly addresses the apparent contradiction that although impaired Hh signaling is the most common pathological mechanism for HPE, the hypo-function of SMO is indeed uncommon. And yet, gain-of-function mutations in SMO are a frequent cause of both basal cell carcinoma and many of the most common pediatric brain cancers.
Many of the complicating factors in precision medicine are evident in the present report on these six cases. Current guidelines (Richards et al., 2015) appropriately rank the relative importance of the novelty of a variant, the de novo status, the co-inheritance of relevant variants, instances of similar variants with proven pathogenicity, the family history, similarly affected relatives, the bioinformatic pathogenicity scores, etc., into an integrated determination. Of the proband-specific findings in this report (Suppl.TableS7), only theFGF8 and SHH mutations meet these criteria for pathogenicity, while the SMO variants remained unclassified. Thus, functional studies are often the determinative factor in assigning clarity to the interpretation. Nevertheless, functional studies are frequently difficult to perform in vivo with developmental programs with an extensive self-regulating GRN.
To date, most published assays of Hh activity have depended on reporter-fusions (e.g. 8X-GLIBS-luciferase) in readily transfectable cells or similar GLI-responsive markers (Myers et al., 2013). While these methods are readily quantifiable, they greatly oversimplify the known tissue interactions of developmental processes that operatein vivo . These cell-based assays often require the absence of Ptch and an intact GRN present in the living animal. A promising advance is the recent development of rapid assays of SMO activity that can determine biological activities in minutes or hours (Myers et al., 2017).
In all of our over-expression assays, we empirically determined that additional interventions were required to obtain interpretable data. Furthermore, the activation, or suppression, of the GRN obscured any differences between the variants themselves, or with the WT control. Indeed, the marker and morphological criteria we used as a biological response required many hours or days to become measurable. We observed that many of these over-expression measures were highly time-dependent (data not shown). By contrast, the ability to generate CRISPER/Cas9 mutant test lines has proven to be an immensely powerful tool. It avoids the requirement for co-interventions that can sometimes obscure the results.
Finally, recent advances have independently supported our identification human p.V404 as an important residue in the 7TM domain of SMO . While formally no longer considered novel (PM2), we observe that the single whole genome detection of p.V404M occurred in a distinct ethnic group for our proband. The analogous murine residue p.V408 is implicated in cholesterol agonist binding (PM1). An engineered variant p.V408F in this site sterically blocks cholesterol and abrogates GLI-luciferase reporter activity (Deshpande et al., 2019). V404, the binding pocket residues for p.V408, was reported the mutation have the potential function as alter sterol affinity or directly stabilize the active SMO conformation (Sharpe et al., 2015). While our human variant p.V404M lacks the same bulky Phe side chain, the structural similarities are consistent with the requirements of this residue being essential inSMO function.
In conclusion, the value of Targeted Sequencing of the Hh pathway has established a framework for integrated genetics interpretations of the potential for gene:gene interactions that can serve as a basis for more comprehensive and accurate genetic counseling in HPE. Strict adherence to existing guidelines indicate that the driver mutations described in this report, such as FGF8 and SHH , rarely require co-morbid modifiers. As whole exome and whole genome data become more readily available for HPE, we anticipate that this autosomal dominant with modifier model will continue to be subject to experimental validation and result in further insight.