Using human sequencing to guide craniofacial research

Abstract

A recent convergence of technological innovations has re-energized the ability to apply genetics to research in human craniofacial development. Next-generation exome and whole genome sequencing have significantly dropped in price, making it relatively trivial to sequence and analyze patients and families with congenital craniofacial anomalies. A concurrent revolution in genome editing with the use of the CRISPR-Cas9 system enables the rapid generation of animal models, including mouse, which can precisely recapitulate human variants. Here, we summarize the choices currently available to the research community. We illustrate this approach with the study of a family with a novel craniofacial syndrome with dominant inheritance pattern. The genomic analysis suggested a causal variant in AMOTL1 which we modeled in mice. We also made a novel deletion allele of Amotl1. Our results indicate that Amotl1 is not required in the mouse for survival to weaning. Mice carrying the variant identified in the human sequencing studies, however, do not survive to weaning in normal ratios. The cause of death is not understood for these mice complicating our conclusions about the pathogenicity in the index patient. Thus, we highlight some of the powerful opportunities and confounding factors confronting current craniofacial genetic research.

Keywords: birth defects; mammal; organism; process, genetics; process, neural crest; process, organogenesis; tissue; tissue, other.

FIGURE 1

A novel congenital malformation syndrome. (a) Pedigree of proband (patient 1). Affected members are shown with the hash marks and the circled members were selected for exome sequencing. (b) The variant in AMOTL1 affects as hightly conserved arginine residue. (c) Sanger sequencing of indicated family members confirms the AMOTL1 sequence variant is present in only patients 1 and 3

FIGURE 2

An allelic series of Amotl1 in the mouse. (a) Wild-type nucleotide sequence of Amotl1 around the desired single nucleotide C>T mutation (blue shaded box). Sequence analysis of Amotl1^R157C mice in this region indicates the desired genome edit was made along with other silent mutations (shown in red) to facilitate CRISPR guide stability and genotyping of modified mice. Amotl1^D1 sequence is shown: (−) indicates the deleted nucleotide. (b) The predicted proteins for wild-type and each of the genome edits. The R157C variant is a single missense codon (red), while the D1 deletion changes codon usage to create 15 missense amino acids (orange) before a stop codon. (c) Sanger sequence validation of the sequence results schematized in (b)

FIGURE 3

Mice carrying the Amotl1^R157C variant are healthy and fertile. Histological analysis (a, b), skeletal preparations (c, d) and whole mount analysis (e, f ) do not reveal any phenotypes which allow us to explain the perinatal lethality in Amotl1^R157C/wt mice as compared to wild-type

FIGURE 4

Cardiac development is normal in Amot1^R157C mice. Whole mount and histological analysis indicates cardiac development is relatively unaffected in Amotl1^R157C/wt mice

FIGURE 5

Amotl1 variants do not significantly affect protein or mRNA levels. (a) Overexpression of AMOTL1^R157C-myc indicates the coding change may allow a post-translational modification which changes the apparent size of the protein. Over a series of experiments, we do not notice a consistent and significant change in protein levels. (b) cDNA RT-PCR analysis shows that the premature stop codon in the Amotl1^D1 allele does not trigger nonsense-mediated decay. Gapdh is shown as a control reaction. (c) Quantititive RT-PCR of five wild-type and five mutant RNA samples from E14.5 head tissue run in triplicate shows no significant reduction in Amotl1 mRNA levels. Data are shown with the mean and upper/lower 95% confidence interval of the mean indicated with horizontal bars