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Application and detection reagent for screening allopurinol tolerant gene methylation marker

An allopurinol and methylation technology, applied in the field of biotechnology, can solve problems such as severe drug eruption in patients, and achieve the effect of improving sensitivity and specificity

Pending Publication Date: 2022-05-10
THE SECOND XIANGYA HOSPITAL OF CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there were still severe drug eruptions in patients with negative screening results and no severe drug eruptions in patients with positive screening results

Method used

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  • Application and detection reagent for screening allopurinol tolerant gene methylation marker
  • Application and detection reagent for screening allopurinol tolerant gene methylation marker
  • Application and detection reagent for screening allopurinol tolerant gene methylation marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Detection of HLA-B*5801 alleles in peripheral blood of allopurinol-induced severe drug eruption patients and allopurinol-resistant patients.

[0045] Genomic DNA was extracted from 2 mL of venous blood from each participant using the QIAamp DNA purification system (Qiagen). The presence or absence of HLA-B*58:01 was detected by the PG5801 DNA detection kit (Pharmigene), and the patients were further divided into four groups: those who carried the HLA-B*58:01 site (25 cases), those who did not Tolerant patients carrying the HLA-B*58:01 site (114 cases), severe drug eruption patients (116 cases) carrying the HLA-B*58:01 site, and those who did not carry the HLA-B*58:01 site Patients with severe drug eruption (8 cases). The kit relies on real-time polymerase chain reaction (PCR) with HLA-B*58:01 sequence-specific primers.

Embodiment 2

[0046] Example 2 Detection of genome-wide DNA methylation levels in peripheral blood of allopurinol-induced severe drug eruption patients and allopurinol-resistant patients.

[0047] Four groups of patients with high DNA quality and matched gender and age were further screened for genome-wide DNA methylation level detection: tolerant patients carrying HLA-B*58:01 sites (18 cases), those without HLA-B *58:01 site tolerance (25 cases), severe drug eruption patients carrying HLA-B*58:01 site (13 cases), severe drug eruption patients not carrying HLA-B*58:01 site ( 6 cases). Extracted DNA samples were subjected to bisulfite conversion using the EZ DNA Methylation™ Kit (Zymo Research) according to the manufacturer's instructions. The transformed DNA was then applied to an Illumina Infinium Methylation Chip (850k). Differentially methylated CpGs were selected according to their beta values ​​using the algorithm in IMA Bioconductor. Differential methylation was defined as an avera...

Embodiment 3

[0048] Example 3 analyzes the application of differentially methylated genes HLA-B, TRAF1 and ITGB2 in the screening of allopurinol-induced severe drug eruption patients and allopurinol-resistant patients.

[0049] The sensitivity and specificity of HLA-B, TRAF1 and ITGB2 methylation levels in screening allopurinol-induced severe drug eruption patients and allopurinol-resistant patients were calculated by ROC curve evaluation statistics. Analysis of tolerant individuals carrying HLA-B*58:01 (18 cases), tolerant individuals not carrying HLA-B*58:01 (25 cases), carrying HLA-B*58:01 The severe drug eruption patients (13 cases) and the severe drug eruption patients (6 cases) who did not carry the HLA-B*58:01 site. It is generally believed that the actual value range of the area under the ROC curve (AUC) is 0.5-1, and the diagnostic value is medium when it is 0.7-0.9, and the diagnostic value is high when it is above 0.9. HLA-B site methylation level distinguishes patients with se...

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Abstract

The invention relates to application and a detection reagent for screening allopurinol tolerant gene methylation markers. The marker comprises an HLA-B * 58: 01 allele, and methylation level detection of HLA-B gene, TRAF1 gene, ITGB2 gene, BCL2 gene, ITGB2-AS1 gene, REPIN1 gene, JPH3 gene, KCNJ1 gene, METTL11B gene and ACSL-1 gene. The method is mainly used for evaluating the risk that a subject suffers from severe drug rash caused by allopurinol, and the sensitivity and specificity of detecting and screening the severe drug rash caused by allopurinol through pure HLA-B * 58: 01 allele are remarkably improved; particularly, the method has important clinical significance on identification of purinol tolerant population with positive HLA-B * 58: 01 allelic genes and intolerant population with negative HLA-B * 58: 01 allelic genes. The method is simple and convenient to operate, high in detection specificity and sensitivity, short in time consumption, small in required sample amount, easy to clinically popularize and wide in application prospect.

Description

technical field [0001] The invention relates to the fields of biotechnology and biomedicine, in particular to an application and a detection reagent for screening allopurinol-resistant gene methylation markers. Background technique [0002] Severe drug eruption (SCAR) is severe drug-induced skin injury with or without other organ damage, which poses a great clinical challenge. Severe drug eruptions mainly include the following types: Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN), and drug reaction with eosinophilia and systemic symptoms (DRESS) (also known as drug-induced hypersensitivity syndrome (DIHS)). The reported incidence of severe drug eruption in hospitalized patients is approximately 2%. Although severe drug eruption is a rare case, its mortality rate can be as high as 50%. Even when patients survive these life-threatening illnesses, they may develop long-term complications such as scarring, visual impairment, interstitial lung disease, chroni...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883
CPCC12Q1/6883C12Q2600/154
Inventor 陆前进赵明刘昱
Owner THE SECOND XIANGYA HOSPITAL OF CENT SOUTH UNIV
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