c.1810C>T polymorphism of NTRK1 gene is associated with reduced survival in neuroblastoma patients

Abstract

Background: TrkA (encoded by NTRK1 gene), the high-affinity tyrosine kinase receptor for neurotrophins, is involved in neural crest cell differentiation. Its expression has been reported to be associated with a favourable prognosis in neuroblastoma. Therefore, the entire coding sequence of NTRK1 gene has been analysed in order to identify mutations and/or polymorphisms which may alter TrkA receptor expression.

Methods: DNA was extracted from neuroblastomas of 55 Polish and 114 Italian patients and from peripheral blood leukocytes of 158 healthy controls. Denaturing High-Performance Liquid Chromatography (DHPLC) and Single-Strand Conformation Polymorphism (SSCP) analysis were used to screen for sequence variants. Genetic changes were confirmed by direct sequencing and correlated with biological and clinical data.

Results: Three previously reported and nine new single nucleotide polymorphisms were detected. c.1810C>T polymorphism present in 8.7% of cases was found to be an independent marker of disease recurrence (OR = 13.3; p = 0.009) associated with lower survival rates (HR = 4.45 p = 0.041). c.1810C>T polymorphism's unfavourable prognostic value was most significant in patients under 18 months of age with no MYCN amplification (HR = 26; p = 0.008). In-silico analysis of the c.1810C>T polymorphism suggests that the substitution of the corresponding amino acid residue within the conservative region of the tyrosine kinase domain might theoretically interfere with the functioning of the TrkA protein.

Conclusions: NTRK1 c.1810C>T polymorphism appears to be a new independent prognostic factor of poor outcome in neuroblastoma, especially in children under 18 months of age with no MYCN amplification.

Figure 1

Sequence variants of the NTRK1 gene identified in the present study. The structure of TrkA receptor. Extracellular part of the protein consists of: two cysteine clusters (Cys); three leucine-rich motifs (LRM) and two immunoglobulin-like C2 domains (Ig-C2). It is followed by a short transmembrane portion (TM), and intracellular juxtamembrane domain (JM) and tyrosine kinase domain (TK). * - newly identified sequence variants, circled - the SNPs having MAF (minor allele frequency) value >5%, arrows - exonic localization of the variants, dishes - intronic localization of the variants.

Figure 2

Homology model of the TrkA TK domain. The model was obtained using rat MuSK kinase structure 1luf [23]. Activation loop (orange) and catalytic loop (blue) are indicated in accordance to de Pablo et al. [31]. The kinase insert fragment of kinase domain constitutes a loop joining two alpha-helices. Most of its residues are missing in the crystal structure presumably due to the loop's high conformational flexibility and therefore not present in homology model (depicted as green beads). The His604 residue (red) is located at the end of structurally determined helical region, distant from ATPase active site (arrow). It may, however, be involved in interactions with residues of the unstructured loop region. Although the function of this loop is not yet known, it may be speculated that it is involved in specific for TrkA interactions with secondary messengers or regulatory proteins. Homology model was visualized with VMD program [32].

Figure 3

Kaplan-Meier estimates of 5-year OS (A) and EFS (B) for NB patients and 5-year OS (C) and EFS (D) for NB patients under 18 months of age and without MYCN amplification with respect to c.1810C>T polymorphism. Figures A and B show results for the entire group of NB patients (n = 145): 133 patients with NTRK1 c.1810CC genotype and 12 cases with NTRK1 c.1810CT/c.1810TT genotype. Figures C and D show results for the group of NB patients under 18 months of age and without MYCN amplification (n = 84). This subgroup consists of 78 patients with NTRK1 c.1810CC genotype and 6 cases with NTRK1 c.1810CT/c.1810TT genotype.