Large-scale sequencing efforts have captured a rapidly growing catalogue of genetic variations. However, the accurate establishment of gene variant pathogenicity remains a central challenge in translating personal genomics information to clinical decisions. Interferon Regulatory Factor 6 (IRF6) gene variants are significant genetic contributors to orofacial clefts. Although approximately three hundred IRF6 gene variants have been documented, their effects on protein functions remain difficult to interpret. Here, we demonstrate the protein functions of human IRF6 missense gene variants could be rapidly assessed in detail by their abilities to rescue the irf6 -/- phenotype in zebrafish through variant mRNA microinjections at the one-cell stage. The results revealed many missense variants previously predicted by traditional statistical and computational tools to be loss-of-function and pathogenic retained partial or full protein function and rescued the zebrafish irf6 -/- periderm rupture phenotype. Through mRNA dosage titration and analysis of the Exome Aggregation Consortium (ExAC) database, IRF6 missense variants were grouped by their abilities to rescue at various dosages into three functional categories: wild type function, reduced function, and complete loss-of-function. This sensitive and specific biological assay was able to address the nuanced functional significances of IRF6 missense gene variants and overcome many limitations faced by current statistical and computational tools in assigning variant protein function and pathogenicity. Furthermore, it unlocked the possibility for characterizing yet undiscovered human IRF6 missense gene variants from orofacial cleft patients, and illustrated a generalizable functional genomics paradigm in personalized medicine.

IMPORTANCE Cutaneous immune-related adverse events (cirAEs) are some of the earliest toxic reactions to emerge following immune-checkpoint inhibitor (ICI) initiation. As an early indicator of robust inflammatory response, cirAEs may be associated with patterns of immune-mediated toxic effects, but associations between these events and noncutaneous immune-related adverse events (irAEs) remain underexplored.OBJECTIVES To characterize patterns of cirAEs and irAEs across care settings and examine associations between the features of first cirAE, overall irAE risk, and risk of specific irAE subtypes. DESIGN, SETTING, AND PARTICIPANTSA retrospective cohort study was conducted at a single academic medical center. The cohort included 358 patients with cancer who initiated anti-programmed death 1/ligand 1 and/or anticytotoxic-T-lymphocyte-4 ICI therapy between January 1, 2016, and March 8, 2019, and developed 1 or more cirAEs, identified using International Statistical Classification of Diseases and Related Health Problems, Tenth Revision codes and confirmed via manual medical record review. All relevant information documented before March 31, 2020, was included.EXPOSURES Anti-programmed death 1/ligand 1 and/or anticytotoxic-T-lymphocyte-4 therapy. MAIN OUTCOMES AND MEASURESAssociations between specific cirAE morphologic classes and patterns of irAEs (occurrence, timeline, organ class, and specific toxic effects). Given the potential that shared underlying factors are associated with the risk of both noncutaneous and cutaneous toxic effects, the presence of observed positive associations between certain cirAE and irAE subtypes was hypothesized. RESULTSOf the 358 patients, 213 were men (59.5%); median age was 65 years (interquartile range, 55-73 years). Nearly half of the patients (177 [49.4%]) with cirAE also developed a noncutaneous irAE. Most patients (128 [72.3%]) experienced their first cirAE before developing any irAE. Several cirAE morphologic classes were found to be associated with overall, organ-based, and specific irAEs. More specifically, mucositis was found to be associated with overall irAE risk (odds ratio [OR], 5.28; 95% CI, 1.11-24.26; P = .04), gastrointestinal irAEs (OR, 5.70; 95% CI,; P = .04), and the specific diagnosis of gastroenterocolitis (OR, 6.80; 95% CI, 1.24-37.39; P = .03). In addition, psoriasis was associated with an increased risk of endocrine irAEs (OR, 4.54; 95% CI, 1.21-17.04; P = .03). CONCLUSIONS AND RELEVANCEIn this cohort study, these findings underscore the risk of multisystem toxic effects in patients experiencing cirAEs and highlight potential opportunities for dermatologists in the management of noncutaneous toxic effects.

Mutations in IRF6, TFAP2A and GRHL3 cause orofacial clefting syndromes in humans. However, Tfap2a and Grhl3 are also required for neurulation in mice. Here, we found that homeostasis of Irf6 is also required for development of the neural tube and associated structures. Over-expression of Irf6 caused exencephaly, a rostral neural tube defect, through suppression of Tfap2a and Grhl3 expression. Conversely, loss of Irf6 function caused a curly tail and coincided with a reduction of Tfap2a and Grhl3 expression in tail tissues. To test whether Irf6 function in neurulation was conserved, we sequenced samples obtained from human cases of spina bifida and anencephaly. We found two likely disease-causing variants in two samples from patients with spina bifida. Overall, these data suggest that the Tfap2a-Irf6-Grhl3 genetic pathway is shared by two embryologically distinct morphogenetic events that previously were considered independent during mammalian development. In addition, these data suggest new candidates to delineate the genetic architecture of neural tube defects and new therapeutic targets to prevent this common birth defect.

Irf6 and Esrp1 are important for palate development across vertebrates. In zebrafish, we found that irf6 regulates the expression of esrp1. We detailed overlapping Irf6 and Esrp1/2 expression in mouse orofacial epithelium. In zebrafish, irf6 and esrp1/2 share expression in periderm, frontonasal ectoderm, and oral epithelium. Genetic disruption of irf6 and esrp1/2 in zebrafish resulted in cleft of the anterior neurocranium. The esrp1/2 mutant also developed cleft of the mouth opening. Lineage tracing of cranial neural crest cells revealed that cleft resulted not from migration defect, but from impaired chondrogenesis. Analysis of aberrant cells within the cleft revealed expression of sox10, col1a1 and irf6 and were adjacent to cells krt4 and krt5 positive. Breeding of mouse Irf6;Esrp1;Esrp2 compound mutants suggested genetic interaction, as the triple homozygote and the Irf6;Esrp1 double homozygote was not observed. Further, Irf6 heterozygosity reduced Esrp1/2 cleft severity. These studies highlight the complementary analysis of Irf6 and Esrp1/2 in mouse and zebrafish and captured a unique aberrant cell population expressing sox10, col1a1 and irf6. Future work characterizing this cell population will yield additional insight into cleft pathogenesis.

Wnt signaling plays a critical role in craniofacial patterning, as well as tooth and bone development. Rspo2 and Rspo3 are key regulators of Wnt signaling. However, their coordinated function and relative requirement in craniofacial development and odontogensis are poorly understood. We showed that in zebrafish rspo2 and rspo3 are both expressed in osteoprogenitors in the embryonic craniofacial skeleton. This is in contrast to mouse development, where Rspo3 is expressed in osteoprogenitors while Rspo2 expression is not observed. In zebrafish, rspo2 and rspo3 are broadly expressed in the pulp, odontoblasts and epithelial crypts. However, in the developing molars of the mouse, Rspo3 is largely expressed in the dental follicle and alveolar mesenchyme while Rspo2 expression is restricted to the tooth germ. While Rspo3 ablation in the mouse is embryonic lethal, zebrafish rspo3-/- mutants are viable with modest decrease in Meckel’s cartilage rostral length. However, compound disruption of rspo3 and rspo2 revealed synergistic roles of these genes in cartilage morphogenesis, fin development, and pharyngeal tooth development. Adult rspo3−/− zebrafish mutants exhibit a dysmorphic cranial skeleton and decreased average tooth number. This study highlights the differential functions of Rspo2 and Rspo3 in dentocranial morphogenesis in zebrafish and in mouse.

Author Contributions: Mr Miller had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis Mr Miller and Ms Ly contributed equally to the study.

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