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Diagnosis

Official Journal of the Society to Improve Diagnosis in Medicine (SIDM)

Editor-in-Chief: Graber, Mark L. / Plebani, Mario

Ed. by Argy, Nicolas / Epner, Paul L. / Lippi, Giuseppe / Singhal, Geeta / McDonald, Kathryn / Singh, Hardeep / Newman-Toker, David

Editorial Board: Basso , Daniela / Crock, Carmel / Croskerry, Pat / Dhaliwal, Gurpreet / Ely, John / Giannitsis, Evangelos / Katus, Hugo A. / Laposata, Michael / Lyratzopoulos, Yoryos / Maude, Jason / Sittig, Dean F. / Sonntag, Oswald / Zwaan, Laura

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2194-802X
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Development and application of a PCR-HRM molecular diagnostic method of SNPs linked with TNF inhibitor efficacy

Mei-juan Yang / Yan-long Hou / Xiao-lan Yang / Chun-xia Wang / Li-xia Zhi / Chong-ge You
  • Corresponding author
  • Department of Clinical Laboratory, Lanzhou University Second Hospital, Lanzhou, No. 82 Cuiyingmen Road, Lanzhou, Gansu, 730000, P.R. China, Phone: +86-0931-8943093
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Published Online: 2018-12-04 | DOI: https://doi.org/10.1515/dx-2018-0062

Abstract

Background

Clinical evidence indicates that genetic variations may interfere with the mechanism of drug action. Recently, it has been reported that the single nucleotide polymorphisms (SNPs) of STAT4, PTPN2, PSORS1C1 and TRAF3IP2RA genes are associated with the clinical efficacy of tumor necrosis factor (TNF) inhibitors in the treatment of rheumatoid arthritis (RA) patients. Therefore, the detection of the SNPs linked with TNF inhibitor efficacy may provide an important basis for the treatment of RA. This study intended to establish molecular diagnostic methods for genotyping the linked SNPs based on high resolution melting (HRM) curve analysis.

Methods

The polymerase chain reaction-HRM (PCR-HRM) curve analysis detecting systems were established by designing the primers of the four SNPs, rs7574865G>T, rs7234029A>G, rs2233945C>A and rs33980500C>T, and the performance and clinical applicability of which were evaluated by using the Sanger sequencing method and genotyping test for 208 clinical samples.

Results

The self-developed molecular diagnostic methods of PCR-HRM were confirmed to be able to correctly genotype the four SNPs, the sensitivity and specificity of which were 100% in this study. The repeatability and reproducibility tests showed that there is little variable in intra-assay and inter-assay (the coefficient of variation ranged from 0.01% to 0.07%). The slight changes of DNA template and primer concentrations, PCR cycle number and reaction system volume had no significant effect on the genotyping performance of the method. The PCR-HRM assays were also applied to other PCR thermocyclers with HRM function and use different saturation fluorescent dyes.

Conclusions

The PCR-HRM genotyping method established in this study can be applied to the routine molecular diagnosis of rs7574865, rs7234029, rs2233945 and rs33980500.

Keywords: genotyping; high resolution melting; single nucleotide polymorphism; TNF inhibitor

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About the article

Received: 2018-08-07

Accepted: 2018-10-03

Published Online: 2018-12-04


Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

Research funding: This work was supported by the Key Research and Development Plan of Gansu Provincial Science and Technology Department (18YF1FA108), Lanzhou University Second Hospital Doctoral Research Fund Project (Ynbskyjj2015-1-6), Hygienic Profession Scientific Research Scheme Management Project of Gansu (GWGL2004-02).

Employment or leadership: None declared.

Honorarium: None declared.

Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.


Citation Information: Diagnosis, 20180062, ISSN (Online) 2194-802X, ISSN (Print) 2194-8011, DOI: https://doi.org/10.1515/dx-2018-0062.

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