Characterisation of the human GFRalpha-3 locus and investigation of the gene in Hirschsprung disease.

BACKGROUND The GDNF family receptor alpha (GFRalpha) proteins are extracellular cell surface bound molecules that act as adapters in binding of the GDNF family of soluble neurotrophic factors to the RET receptor. These molecules are essential for development of many neural crest derived cell types and the kidney. Mutations in RET and in two members of the GDNF ligand family are associated with Hirschsprung disease (HSCR), a congenital absence of the enteric ganglia. Members of the GFRalpha family are also candidates for HSCR mutations. One such gene is GFRalpha-3, which is expressed in the peripheral nervous system and developing nerves. OBJECTIVE We have characterised the structure of the human GFRalpha-3 locus and investigated the gene for sequence variants in a panel of HSCR patients. METHODS Long range PCR or subcloning of PAC clones was used to investigate GFRalpha-3 intron-exon boundaries. A combination of single strand conformation polymorphism (SSCP) analysis and direct sequencing was used to investigate GFRalpha-3 sequence variants. RESULTS GFRalpha-3 spans eight coding exons and has a gene structure and organisation similar to that of GFRalpha-1. We identified three polymorphic variants in GFRalpha-3 in a normal control population, a subset of which also occurred in HSCR patients. We did not detect any sequence variants within the coding sequence of GFRalpha-3. We found a base substitution in the 5' UTR of GFRalpha-3, 15 base pairs upstream of the translation start site. A second substitution was identified in intron 4 (IVS4-30G>A) between the splice branch site and the splice acceptor site. The final variant was a 2 base pair insertion within the splice donor consensus sequence of exon 7 (IVS7+4ins GG). CONCLUSIONS We did not detect any correlation between variants of GFRalpha-3 and the HSCR phenotype. Our data suggest that mutations of this gene are not a cause of HSCR.