A Premature Stop Codon in RAF1 Is the Priority Candidate Causative Mutation of the Inherited Chicken Wingless-2 Developmental Syndrome

Abstract

The chicken wingless-2 (wg-2) mutation is inherited in an autosomal recessive fashion, and the resulting phenotype in mutant (wg-2/wg-2) individuals is a developmental syndrome characterized by absent wings, truncated legs, craniofacial as well as skin and feather defects, and kidney malformations. Mapping and genotyping established that the mutation resides within 227 kilobases (kb) of chromosome 12 in a wg-2 congenic inbred line. A capture array was designed to target and sequence the candidate region along with flanking DNA in 24 birds from the line. Many point mutations and insertions or deletions were identified, and analysis of the linked variants indicated a point mutation predicted to cause a premature stop codon in the RAF1 gene. Expression studies were conducted inclusive of all genes in the candidate region. Interestingly, RAF1 transcription was elevated, yet the protein was absent in the mutants relative to normal individuals. RAF1 encodes a protein integral to the Ras/Raf/MAPK signaling pathway controlling cellular proliferation, and notably, human RASopathies are developmental syndromes caused by germline mutations in genes of this pathway. Our work indicates RAF1 as the priority candidate causative gene for wg-2 and provides a new animal model to study an important signaling pathway implicated in limb development, as well as RASopathies.

RAF1 deficiency causes a lethal syndrome that underscores RTK signaling during embryogenesis

“…Wojnowski et al ( 1998 ) noted thinner and less differentiated dermal and epidermal layers in Raf1 −/− mice, while Yamaguchi et al ( 2004 ) found that heart‐specific Raf1 −/− mice exhibited cardiac dysfunction and apoptosis. More recently, the recessive‐acting wingless2 mutation found in Gallus gallus —which results in a phenotype strikingly similar to this syndrome with truncated limbs, craniofacial, cardiac, and skin/feather defects—was linked to a homozygous premature stop codon in Raf1 (Youngworth & Delany, 2019 ).…”

A Progeroid Syndrome Caused by RAF1 deficiency Underscores the importance of RTK signaling for Human Development

“…10 More recently, the recessive-acting wingless2 mutation found in Gallus galluswhich results in a phenotype strikingly similar to this syndrome with truncated limbs, craniofacial, cardiac and skin/feather defectswas linked to a homozygous premature stop codon in Raf1. 37 These observations lead us to conjecture that the Mendelian disease and by inference ACFS may be caused by impaired RAF1 signaling. Hence, it will be worthwhile checking for mutations in RAF1, its upstream regulators and downstream effectors in previously reported ACFS cases.…”

“…10 More recently, the recessive-acting wingless2 mutation found in Gallus gallus – which results in a phenotype strikingly similar to this syndrome with truncated limbs, craniofacial, cardiac and skin/feather defects – was linked to a homozygous premature stop codon in Raf1 . 37…”