Scientists at Nationwide Children’s Hospital in Ohio and the Van Andel Research Institute in Michigan have demonstrated the first successful correction of limb length in a mouse model of FZD2-associated autosomal dominant Robinow Syndrome. The findings, published on February 15 in the journal Development, “Successful therapeutic intervention in new mouse models of frizzled 2-associated congenital malformations,” opens the door for new therapeutic interventions.
Robinow Syndrome is a set of genetic disorders that affects the growth and development of the skeletal system, resulting in facial abnormalities, cleft palate, and shortened limbs. Although autosomal dominant Robinow Syndrome disorders are extremely rare, they’re associated with mutations that can be inherited from a single parent (dominant mutations) or arise spontaneously.
Senior author of the study, Rolf Stottmann, PhD, scientist in the Institute for Genomic Medicine at Nationwide Children’s Hospital and an associate professor at The Ohio State University College of Medicine said, “We began the project by studying the genomes of families with structural birth differences of the brain and face who had not yet received a genetic diagnosis. We identified that one of the initial families in this cohort had a mutation in the FZD2 gene.”
In an earlier study the researchers had identified a pathogenic human FZD2 variant in individuals with FZD2-associated autosomal dominant Robinow syndrome. The variant encoded a protein with a premature stop and loss of 17 amino acids, including a region of the consensus binding sequence for disheveled—a key component of developmental Wnt signaling. FZD2, (Frizzled 2) encodes a transmembrane Wnt receptor involved in sending signals that cells use to organize themselves into tissues.
“To model this variant, we used zygote microinjection and i-GONADbased CRISPR/Cas9-mediated genome editing to generate a mouse allelic series,” the authors noted.
Stottmann and colleagues used CRISPR/Cas9 genome-editing to induce mutations in mice Fzd2 that recapitulate mutations found in humans with FZD2-associated autosomal dominant Robinow Syndrome. Mice with these Fzd2 mutations exhibited facial and skeletal malformations including cleft palates and limbs less than half the normal size, resembling disease features seen in patients.
The authors also generated two germline mouse alleles with small deletions: Fzd2D3 and Fzd2D4. Homozygotes for each of these alleles also exhibited a highly penetrant cleft palate phenotype, shortened limbs and perinatal lethality.
“The in vivo replication represents an approach for further investigating the mechanism of FZD2 phenotypes and demonstrates the utility of CRISPR knock-in mice as a tool for investigating the pathogenicity of human genetic variants,” the authors noted.
To rescue the missing signals, the scientists intervened by treating pregnant mice with a drug that stimulates the Wnt signaling pathway.
“We reasoned that the action of the IIIC3a molecule as an antagonist of the Wnt antagonist DKK may act to augment the pathway and partially rescue the genetic lesions in Fzd2,” the authors noted.
Indeed, in utero treatment with the DKK inhibitor IIC3a, normalized the length of limbs in mouse embryos homozygous for the Fzd2D4 mutation.
“This drug is an attractive option because we think we know how it works and previous work had shown that it could rescue cleft palates in a mouse model,” said Stottmann. “The idea of treating the limb bones medically rather than surgically is a really important proof-of-principle, which we demonstrate in this study. We are very excited to test if this could work in the context of other genes associated with autosomal dominant Robinow Syndrome.”