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

1.我们对23例日本患者和8例外国患者的TrkA基因进行了分析，发现这些患者都存在致病突变。我们还报告了日本CIPA家系中基因内多态位点的特征，并描述了这些位点上等位基因的单倍型关联。超过50%的CIPA染色体具有我们前面描述的移码突变(R548fs)。该突变明显表现出与正常染色体中罕见单倍型的连锁不平衡，强烈提示它是一种共同的创始人突变。单亲二体(UPD)的定义是在二倍体个体中只有一个患者的一对染色体的存在。我们观察到一例非孟德尔遗传的男性CIPA患者。TrkA基因座的单倍型分析和1号染色体全染色体的等位基因分析表明，染色体…更多的配对完全是从他父亲那里遗传来的。通过对1号染色体以外的常染色体的分析，排除了非母体。因此，我们确定1号染色体的一个完整的父系同体是导致TrkA基因突变减少到纯合的原因，导致CIPA。通过外显子陷阱分析，我们已经证明了TrkA基因的内含子分支位点(IVS7-33T&GT；A)突变在体外会导致异常剪接。我们还报告了来自不同民族的32个CIPA家系中的11个推测的错义突变。在这里，我们将相应的突变引入到TrkA的cDNA中，并检测了NGF刺激的自磷酸化。胞外区的两个突变体(L93P和L213P)在神经细胞中发生异常加工，表现出自磷酸化作用减弱。酪氨酸激酶区的5个突变体(G516R、G571R、R643W、R648C和G708S)被处理为野生型TrkA，但神经细胞和非神经细胞的自磷酸化显著降低。相反，R85S和H598Y；G607V以前被检测为双突变和三突变，可能是特定种族背景下的多态性。另一个可能的突变体D668Y可能是一种罕见的多态，或者可能在不影响自身磷酸化的情况下削弱TrkA的功能。酪氨酸激酶结构域的突变残基在多种受体酪氨酸激酶(RTK)中都是保守的，可能与这些蛋白的关键功能有关。因此，自然发生的带有功能丧失的TrkA错义突变为RTK家族的结构-功能关系提供了相当多的洞察力。较少

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