Using human sequencing to guide craniofacial research

Abstract

Summary 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.

De novo variant in AMOTL1 in infant with cleft lip and palate, imperforate anus and dysmorphic features

“…AMOTs, in particular, AMOTL1, are involved in regulation of organogenesis. Similar to the family reported by Liegel et al (2019), the individual described herein harbors a heterozygous missense variant in AMOTL1 that is predicted to alter a highly conserved site. The proband suffered from oro-palatine and earlobe maldevelopment.…”

“…Notably, the missense variant reported in Liegel et al (2019) affects a residue only three amino acids away from that described here (p. (Arg157Cys) and p.(Pro160Leu), respectively), suggesting that there may be an important functional domain in this region. Although studies in mice with a missense Arg157Cys AMOTL1 variant were shown to result in increased embryonal death rates in both heterozygous and homozygous forms, none of the phenotypic features seen in humans with similar mutations were observed.…”

“…The shared phenotypic features between our patient and the family described by Liegel et al (2019) (Table 1).…”

“…Although studies in mice with a missense Arg157Cys AMOTL1 variant were shown to result in increased embryonal death rates in both heterozygous and homozygous forms, none of the phenotypic features seen in humans with similar mutations were observed. Liegel et al suggested that humans represented the milder effect of such variants, and that the pathology may result from a neomorphic allele acting in a dominant negative fashion (Liegel et al, 2019). Considering the low pLI score of the gene (pLI = 0) and the o/e ratio (0.3), haploinsufficiency is not a likely mechanism (Karczewski et al, 2020), urging caution of AMOTL1 variant interpretation.…”

Embracing human genetics: a primer for developmental biologists

“…In fact, it is now accepted that the previous thresholds for calling a variant rare (and therefore potentially deleterious) were too lenient and may account for many published false associations between variants and human disease (Lek et al, 2016). In the absence of independent cases, detailed functional studies in model systems are essential for providing the evidence of causality (Liegel et al, 2019;Lukacs et al, 2019).…”