Molecular pathology of congenital insensitivity to pain with anhidrosis due to genetic defects of the receptor tyrosine kinase for nerve growth factor

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1. We have analyzed the TRKA gene derived from 23 Japanese and 8 foreign patients with congenital insensitivity to pain with anhidrosis (CIPA) and detected responsible mutations in all these patients. We also report the characterization of intragenic polymorphic sites and describe the haplotypic associations of alleles at these sites in Japanese CIPA families. More than 50% of CIPA chromosomes share the frameshift mutation (R548fs) that we described earlier. This mutation apparently shows linkage disequilibrium with a rare haplotype in normal chromosomes, strongly suggesting that it is a common founder mutation.2. Uniparental disomy (UPD) is defined as the presence of a chromosome pair that derives from only one patient in a diploid individual. We have observed a male CIPA patient with non-Mendelian inheritance. He had a homozygous mutation at the TRKA locus on chromosome 1. Haplotype analysis of the TRKA locus and allelotype analyses of whole chromosome 1 revealed that the chromosome … More pair was exclusively derived from his father. Non-maternity was excluded by analyses of autosomes other than chromosome 1. Thus, we have identified a complete paternal isodisomy for chromosome 1 as the cause of reduction to homozygosity of the TRKA gene mutation, leading to CIPA.3. We have demonstrated that an intronic branch-site (IVS7-33T>A) mutation in the TRKA gene causes aberrant splicing in vitro by the exon-trap analysis. We also reported 11 putative missense mutations in 32 CIPA families from various ethnic groups. Here we have introduced the corresponding mutations into the TRKA cDNA and examined NGF-stimulated autophosphorylation. Two mutants (L93P and L213P) in the extracellular domain were aberrantly processed and showed diminished autophosphorylation in neuronal cells. Five mutants (G516R, G571R, R643W, R648C and G708S) in the tyrosine kinase domain were processed as wild-type TRKA but showed significantly diminished autophosphorylation in both neuronal and non-neuronal cells. In contrast, R85S and H598Y; G607V detected previously as double and triple mutations, are probably polymorphisms in a particular ethnic background. The other putative mutant D668Y might be a rare polymorphism or might impair the function of TRKA without compromising autophosphorylation. Mutated residues in the tyrosine kinase domain are conserved in various receptor tyrosine kinases (RTKs) and probably contribute to critical function of these proteins. Thus, naturally occurring TRKA missense mutations with loss-of-function provide considerable insight into the structure-function relationship in the RTK family. Less