NTRK1 Affected Pain: Mutations In Congenital Insensitivity To Pain With Anhidrosis And Polymorphisms In Human Pain Sensitivity

Part Ⅰ NTRK1 mutations and the rare phenotype "painless and anhidrosis"ObjectiveThis study investigated phenotypes of congenital insensitivity to pain with anhidrosis, screened genotypes of NTRK1 and NGF, and analyzed properties of NTRK1 mutations for the next project involving associations of the NTRK1 SNPs with pain.MethodsPatients affected congenital insensitivity to pain with anhidrosis were enrolled. DNA from patients and their parents was extracted from peripheral blood.Seventeen exons with at least 50 nucleotides encompassing intron-exon boundaries in NTRK1 and three in NGF were amplified by PCR. Amplification products were purified and sequenced on both strands and the results were analyzed in Chromas version 2.22. Mutations identified were named according to Human Genome Variation Society guidelines, and were matched against data in dbSNP, dbVar and ClinVar by using Variation Reporter at NCBI (http://www.ncbi.nlm.nih.gov/variation/tools/reporter/). Frameshift mutations were interpreted based on NCBI annotation of the genome, as well as on protein sequence and functional information in UniProt (http://www.uniprot.org). Missense mutations were characterized by location in the protein, amino acid substitution score, and conservation of the mutated residue. PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) was also used to predict the impact of missense mutations. Mutations deemed to be pathogenic were screened by using ligase detection reactions (LDRs) against a control cohort of 1,007 unaffected Chinese.ResultsThirteen patients from eleven unrelated families were enrolled. All target segments were successfully amplified. Tenmutations were considered pathogenic, including a frameshift mutation,a nonsense mutation, six missense mutations, and two intron mutations. All of these ten pathogenic mutations are concentrated in the catalytic domain and NGF-binding region. Both C.1804C>T and c.963delG are possibly the founder mutations in Chinese patients with congenital insensitivity to pain with anhidrosis.ConclusionsThe frequency of NTRK1 mutations is significantly higher than that of NGF, which indicates that NTRK1 is attractive for investigating the effects of polymorphisms. Also, the mutations of NTRK1 are concentrated in the catalytic domain and NGF-binding region, which would facilitate the association analysis between the pain individual difference and the single nucleotide polymorphism. The ethic features of mutations in NTRK1 in patients with congenital insensitivity to pain with anhidrosis need to be further explored for determining whether the associations of the genotypes with phenotypes exist and improving insights of the structures and the functions ofNTRK1.Part Ⅱ NTRK1 polymorphisms and human pain sensitivityObjective The goal of this study was to assess the correlations between NTRK1 SNPs and pain individual differences.MethodsHealthy female undergraduates were enrolled at Tongji Medical College, Huazhong University of Science and Technology. Blood (3-5 mL) was collected from the elbow vein. Genomic DNA was extracted from blood samples. The tag SNP selection was based on phase 3 data of the HapMap CHB reference population database and was performed using the Tagger program included in the Haploview 4.2 software. The aim was to capture all SNPs with a minor allele frequency of greater than 5% in the HapMap database by setting the limit for the pairwise r2 to greater than or equal to 8. Genotyping of the NTRK1 SNPs for all participants was performed by using ligase detection reactions (LDRs).All genetic association analyses were conducted using PLINK, version 1.07 (http://pngu.mgh.harvard.edu/purcell/plink/). Summary statistics were calculated, and the entire sample was tested to determine whether the null hypothesis of the Hardy-Weinberg equilibrium (HWE) could be rejected by applying the χ2 method. SNPs showing deviation from HWE (p< 0.05) were excluded from the final analysis.Associations between the NTRK1 SNPs and pain perception were examined using linear regression analysis. An additive model (each copy of the minor allele having an effect) was considered. The experimental pain variables were analyzed here. Association analyses were performed using variables including age and body mass index (BMI) after adjustment for potential confounding factors. The pvalue less than0.01 was viewed as significant to balance the need for control of type I error and limit the chances of type II error. The other statistical associations (0.011 <p<0.05) were viewed as being of nonsignificant trends.Demoraphic analysis, independent-sample t-test, and analysis of variance of quantitative traits were conducted using SPSS. All analyses used the maximum number of cases available for each phenotype. A 2-tailed probability value of p< 0.05 was used as the criterion for statistical significance.ResultsThree hundred and nine healthy female undergraduates were included in the analysis and 13 tagSNPs of NTRK1 were genotyped. The associations between SNPs of NTRK1 and pain perception were analyzed. SNPs rs2644604, rs6334 and rs1800880 showed statistically significant positive association with S-PTO. SNP rs4661229 showed negative association with QPT, as rs10908521 with WLT. Additionally, a possible relevance without statistical significance were showed between rs11264577 and D-PPT, rs 10908521 and S-PPT, rs56252149 and QPT, as well rs10908521, rs7516736 and WLT, rs6689750 and WLT.ConclusionsNTRK1 SNPs showed statistically significant associations with pain perception in females and they might be one genetic factor contributing to pain individual indifference. NTRK1 SNPs have different effects on different pain, which improve insights of the function of NTRK1 and the signaling pathways for pain. NTRK1 is required for individual pain treatment and developing pain gene chips.