Development and application of a PCR-HRM molecular diagnostic method of SNPs linked with TNF inhibitor efficacy
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Mei-juan Yang
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.
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.
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©2019 Walter de Gruyter GmbH, Berlin/Boston
Artikel in diesem Heft
- Frontmatter
- Editorial
- The Model List of Essential In Vitro Diagnostics: nuisance or opportunity?
- Reviews
- Skeletal myopathies as a non-cardiac cause of elevations of cardiac troponin concentrations
- Histo-genomics: digital pathology at the forefront of precision medicine
- Opinion Papers
- Patients as diagnostic collaborators: sharing visit notes to promote accuracy and safety
- Understanding the “philosophy” of laboratory hemostasis
- Original Articles
- Serious misdiagnosis-related harms in malpractice claims: The “Big Three” – vascular events, infections, and cancers
- Electronic health records, communication, and data sharing: challenges and opportunities for improving the diagnostic process
- Mortality and morbidity rounds (MMR) in pathology: relative contribution of cognitive bias vs. systems failures to diagnostic error
- Experienced physician descriptions of intuition in clinical reasoning: a typology
- A randomized experimental study to assess the effect of language on medical students’ anxiety due to uncertainty
- Development and application of a PCR-HRM molecular diagnostic method of SNPs linked with TNF inhibitor efficacy
- Acute rejection in kidney transplantation and the evaluation of associated polymorphisms (SNPs): the importance of sample size
- Letter to the Editor
- Physically damaged second victims by diagnostic delay in pulmonary tuberculosis of hospitalized patients. Cognitive errors with urgent need for prevention
- The physical examination, including point of care technology, is an important part of the diagnostic process and should be included in educational interventions to improve clinical reasoning
- Case Reports
- Case reports of diagnostic error: liposarcoma mistaken for hematoma in an obese female with concurrent ipsilateral thrombosis on rivaroxaban
- Macro-CK type 2 in metastatic prostate cancer
Artikel in diesem Heft
- Frontmatter
- Editorial
- The Model List of Essential In Vitro Diagnostics: nuisance or opportunity?
- Reviews
- Skeletal myopathies as a non-cardiac cause of elevations of cardiac troponin concentrations
- Histo-genomics: digital pathology at the forefront of precision medicine
- Opinion Papers
- Patients as diagnostic collaborators: sharing visit notes to promote accuracy and safety
- Understanding the “philosophy” of laboratory hemostasis
- Original Articles
- Serious misdiagnosis-related harms in malpractice claims: The “Big Three” – vascular events, infections, and cancers
- Electronic health records, communication, and data sharing: challenges and opportunities for improving the diagnostic process
- Mortality and morbidity rounds (MMR) in pathology: relative contribution of cognitive bias vs. systems failures to diagnostic error
- Experienced physician descriptions of intuition in clinical reasoning: a typology
- A randomized experimental study to assess the effect of language on medical students’ anxiety due to uncertainty
- Development and application of a PCR-HRM molecular diagnostic method of SNPs linked with TNF inhibitor efficacy
- Acute rejection in kidney transplantation and the evaluation of associated polymorphisms (SNPs): the importance of sample size
- Letter to the Editor
- Physically damaged second victims by diagnostic delay in pulmonary tuberculosis of hospitalized patients. Cognitive errors with urgent need for prevention
- The physical examination, including point of care technology, is an important part of the diagnostic process and should be included in educational interventions to improve clinical reasoning
- Case Reports
- Case reports of diagnostic error: liposarcoma mistaken for hematoma in an obese female with concurrent ipsilateral thrombosis on rivaroxaban
- Macro-CK type 2 in metastatic prostate cancer
