Molecular Analysis of Gli3, Ihh, Rab23, and Jag1 in a Rabbit Model of Craniosynostosis

Abstract

These data indicate that the causal locus for heritable craniosynostosis in this rabbit model is not located within the protein coding regions of Gli3, Ihh, Rab23, or Jag1.

Molecular Analyses in a Rabbit Model of Craniosynostosis: Likely Exclusion of Known Candidate Genes as the Loci of Origin

“…Sequencing of known candidate genes is necessary due to the limited availability of commercial tools designed for genetic characterization of the rabbit. To date we have excluded Fgfr1 , Fgfr2 , Fgfr3 , Tgfbr1 , Tgfbr2 , Twist1 , Efna4 , Efnb1 , Gli3 , Ihh , Jag1 , and Rab23 as likely causal loci within the CS rabbit (Gallo et al, 2013; Gallo et al, 2014; Gilbert et al, 2018; Taylor et al, 2018). This study examines the vast majority of the remaining candidates that may reasonably be anticipated to contribute to the synostotic phenotype in the rabbit.…”

“…Synostotic (n = 4) and wild-type (n = 4) animal groupings used in this study have previously been described (Gilbert et al, 2018; Taylor et al, 2018). Briefly, the CS rabbits exhibit an early-onset phenotype with bilateral fusion of the coronal suture by 10 days of age.…”

“…The methods used for tissue collection, RNA isolation, and complementary DNA (cDNA) preparation have previously been described (Gilbert et al, 2018; Taylor et al, 2018). Briefly, total RNA was isolated from ear snips (∼50 mg tissue) using the RNAqueous-Micro Total RNA Isolation Kit (ThermoFisher Scientific, Waltham, Massachusetts), and cDNA was prepared using the AccuScript High Fidelity 1st Strand cDNA Synthesis Kit (Fisher Scientific, Pittsburgh, Pennsylvania).…”

“…Polymerase chain reaction amplification was performed using conditions as previously described by our laboratory (Gilbert et al, 2018; Taylor et al, 2018). Polymerase chain reaction products were purified using the Silica Bead DNA Gel Extraction Kit (ThermoFisher Scientific) according to the manufacturer’s protocol.…”

“…We have bred this individual and her offspring and have developed a breeding colony of rabbits with familial, primary coronal suture synostosis (Mooney et al, 1994a; Mooney et al, 1994b). The genetic basis for CS in this naturally occurring animal model remains uncertain although we have excluded a panel of likely candidates including Fgfr1 , Fgfr2 , Fgfr3 , Tgfbr1 , Tgfbr2 , Twist1 , Efna4 , Efnb1 , Gli3 , Ihh , Jag1 , and Rab23 (Gallo et al, 2013; Gallo et al, 2014; Gilbert et al, 2018; Taylor et al, 2018). Other candidates who are recurrently linked to fusion of the coronal suture in patients include a panel of developmentally regulated transcription factors ( Erf , Msx2 , Tcf12 , Zeb2 ), rare mutations affecting genes involved in development and metabolism ( Cyp26b1 , Il11ra , Por , Ski ), and genes associated with tissue mineralization ( Alpl , Ostm1 ; Table 1; Heuzé et al, 2014).…”