Novel Stem Cell Model of Frontonasal Dysplasia derived from Pedigree with ALX1-Mutation
Janina Kueper, MD1,2, Jonathan Pini, Ph.D.1,2, Yiyuan D. Hu, BS1,2, Nikhil Sobti, BS3, Megan Rebello, BS1,2, Kana Ishii, Ph.D.4, Nikkola Carmichael, M.Sc.5, Victoria Perroni6, Richard L. Maas, MD, PhD6, Yevgenya Grinblatt, PhD7, Eric C. Liao, MD, PhD1,2.
1Massachusetts General Hospital/Center for Regenerative Medicine, Boston, MA, USA, 2Shriners Hospital for Children/Division of Plastic and Reconstructive Surgery, Boston, MA, USA, 3Boston University, Boston, MA, USA, 4Hokkaido University, Hokkaido, Japan, 5Brigham and Women's Hospital, Boston, MA, USA, 6Brigham and Women's Hospital, Boston, MA, USA, 7University of Wisconsin-Madison, Boston, MA, USA.
PURPOSE: Frontonasal dysplasia (FND) is characterized by developmental abnormalities of the midface caused by a disruption of the fusion of the frontonasal with the paired maxillary prominences. Consequent facial malformations are debilitating and may include ocular defects, oblique facial clefts of the soft and hard palate, alveolus, nose and upper lip. Mutations of ALX1 homeodomain transcription factor have recently been associated with FND in both animal models and humans. However, the molecular and developmental mechanisms that underlie FND remain poorly defined.
METHODS: We performed Whole Exome Sequencing (WES) on 4 children affected with FND of 13 born from consanguineous parents and uncovered pathogenic ALX1 gene variant L165F. We utilized CRISPR-Cas9 gene editing in zebrafish to create single and compound mutants of ALX1, ALX3, and ALX4a/b. Additionally, we generated induced Pluripotent Stem Cells (iPSC's) from the unaffected parent, sibling and ALX1-/- subjects. We formed Embryoid Bodies from the iPSC's to investigate the expression of markers of craniofacial and eye development in order to understand the potentially shared role of ALX1 in both developmental processes.
RESULTS: We identified a missense L165F variant in the homeodomain of ALX1 leading to a loss-of-function of this transcription factor. This mutation was found to be heterozygous in the parents (ALX1+/-), wildtype in the unaffected sibling, and homozygous in the children born with FND (ALX1-/-). Using CRISPR, we generated mutant alx1-/- zebrafish which exhibited reduced gene expression and anomalies of the craniofacial cartilage of the median palate, corresponding to the frontonasal oblique facial cleft phenotype of the subjects. The alx1-/- zebrafish also formed smaller Meckel's cartilage, analogous to human micrognathia. We also found that ALX1 mutant zebrafish displayed strong increases in the expression of ALX3 and ALX4a at early developmental stages relative to their wild-type counterparts, likely related to a redundancy of these genes. The iPSC's developed from the patient's samples developed as well as those of the father and a control, pointing toward a rule in later development of ALX1. Through the analysis of the EB's developed from the iPSC's, we found that ALX1 loss of function resulted in decreased expression of FOXD3 and SOX10, genes relevant to neural crest differentiation, as well as PAX6 and its target gene SIX6, both key genes of eye development.
We found that ALX1 plays a vital role in the development of both facial and ocular structures, through regulation of cranial neural crest progenitors that contribute to facial and eye structures of the frontonasal process. Identifying the genetic basis of developmental disorders such as FND allows for both a deeper understanding of molecular processes that regulate midface development. This work also highlights the translation of surgical care to mechanistic discovery through the application of stem cell and CRISPR gene editing approaches, underscoring the unique advantage of the craniofacial surgeon-scientists.
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