I. Study Object
(i) Into the group strategy: continuous into group.
(ii) Grouping strategy：
Case group.
Inclusion criteria：
A clear history of acetaminophen (or acetaminophen-containing drugs) ingestion.
Plasma and/or urine testing for acetaminophen components if history of
ingestion is unclear.
Monitoring of Alanine aminotransferase (ALT) or Aspartate Aminotransferase
(AST) ≥ 1000 IU/L at any time after APAP administration and Roussel Causality
Assessment Method(RUCAM) score > 6
Age ≥ 14 years old.
The subject or guardian agrees to participate in this project and signs an
informed consent form.
Exclusion criteria： •The use of drugs for which frequency of adverse reactions to
liver damage is defined as "common or very common" (≥1%) in the instructions.
Concurrent use of herbs that are clearly susceptible to liver damage (see list
of definitions in the Annex).
Have a known definite cause of liver damage: active viral hepatitis; alcoholic
liver disease; autoimmune liver disease; primary or secondary liver tumors; and
other underlying liver disease that has affected liver function.
Those who fail to provide complete general information and clinical
information.
Subjects or guardians who do not agree to see this project do not sign the
informed consent form.
Control group
Inclusion criteria：
A clear history of acetaminophen (or acetaminophen-containing drugs) ingestion.
Plasma and/or urine testing for acetaminophen components if history of APAP
ingestion is unclear.
Age: ≥ 14 years old.
The subject or guardian agrees to participate in this project and signs an
informed consent form.
Exclusion criteria： •The use of drugs for which frequency of adverse reactions to
liver damage is defined as "common or very common" (≥1%) in the instructions.
•Concurrent use of herbs that are clearly susceptible to liver damage (see list of
definitions in the Annex).
•There are known definite causes of liver damage (see attached list of definitions):
active viral hepatitis; alcoholic liver disease; autoimmune liver disease; primary
or secondary liver tumors; and other underlying liver disease that has affected
liver function.
•Those who fail to provide complete general information and clinical information.
•Subjects or guardians who do not agree to see this project do not sign the informed
consent form.
(iii) Matching strategy.
Matching principle：
•Case and control participants were matched according to ingested dose, duration of
antidote administration, and duration of gastric lavage.
•1:2 matching.
Confounding factors：
•Dose intake: patient report (primary) + blood concentration test (secondary)
•Antidote use time: <4 hours, 4-24 hours, >24 hours
•Gastric lavage time: <1 hour, ≥1 hour
(iv) Estimation of sample size
Parameter source：
Focus on drug metabolizing enzymes, immunogenic loci
The distribution of APAP-dependent DILI susceptibility allele frequencies in
Asian populations based on literature findings.
Parameter value：
Hypothesis: Gene-Environment.
Outcome Model: Baseline risk P0=0.10.
Genetic Effect =1.2,
Power=0.8, Type I error rate=0.05 (two-sided).
Results： The case sample size should be 113 with a control sample size of 226.
II. Exposure/risk factors.
(i) Definition. Exposure factors refer to susceptibility genes for DILI caused by APAP,
including：
(ii) Measurement methods.
Adopt candidate gene strategy. The candidate gene strategy is a flexible approach
with low cost, relatively simple quality control and statistical analysis, and a
relatively easy biological explanation behind the association because the genes
selected are all important for the disease.
Using the hypothesis-driven approach. Considering the hypothesis that some specific
genes may be associated with the study outcome, a sequence-based approach was chosen
for the analysis of potentially functional single nucleotide polymorphism (SNPs),
selecting SNPs that are more likely to be of functional value associated with drug
metabolizing enzymes, immunogenic loci, drug transport proteins, drug-acting
receptors and other links, especially those located in candidate genes coding
regions.
Using custom chips. The Expanded Multi-Ethnic Genotyping Array (MEGAEX), a
microarray built by consortia with enhanced capabilities to understand complex
diseases in a variety of populations. This commercially available microarray
determines individual drug response through the detection of 2,036,060 marker loci.
It also complements HLA-A and HLA-B immune loci information to complete
Genotype-Imputation.
III. Quality control The study has a strict standard test operation procedure, and
relevant training is conducted for the personnel involved in the test before the start of
the experiment, and the test can only be conducted after passing the training. We have a
quality control center with dedicated personnel responsible for subject progress and data
quality control. An online digital randomization platform and information entry
management system is set up based on the web server terminal, which is capable of timely
case randomization grouping and electronic clinical case observation form information
entry, and can effectively and automatically check and correct errors during the
information entry process. That is, it helps to ensure the accuracy and reliability of
the entered information, and can provide real-time data monitoring for the supervisors.
In addition, this study will monitor the conduct of experiments and data entry.
IV. Data Management Plan
Data Center: The data management center established by the national data management
standard undertakes data management work.
Data management system: REDCap is used to establish electronic case report forms for
online data collection and management.
Data collection: Data entry by independent data registrants not involved in the
study in the study lead unit.
Data consistency monitoring: Data consistency monitoring by an independent
third-party monitor.
Data audit and locking: Data is audited, cleaned and locked by a dedicated data
manager.
V. Statistical analysis plan
(i) Hypothesis testing. H0: No difference in genes between subjects with and without
liver damage after ingestion of APAP H1: Significant genetic differences between subjects
with and without liver damage after ingestion of APAP
(ii) Analytical strategies.
Continuous variables were described as mean ± standard deviation (SD) or median of
interquartile range (IQR), and differences between groups were analyzed by
two-factor ANOVA or nonparametric tests. Categorical variables were expressed as
numbers and percentages and analyzed by logistic regression models.
Hardy-Weinberg equilibrium (HWE) between control groups was tested using the
goodness-of-fit χ2 test. Using haplotype analysis view（Haploview） 4.2 software, the
haplotype region group was selected considering the linkage disequilibrium between
tags of single nucleotide polymorphisms (tagSNPs).
Multivariate conditional logistic regression analysis was performed to estimate the
association between genotype and risk of DILI from APAP by dominance ratios (ORs)
and 95% confidence intervals (CIs) with gastric lavage and antidote use as
covariates.
Three different genetic models were used to synthesize the role of tagSNPs. Software
package for Hardy-weinberg analysis(SHEsis) online program was used for haplotype
analysis under logistic regression model.
All analyses were performed using Statistics is a powerful statistical software
platform(SPSS) for Windows (version 26.0 USA). Two-tailed P values <0.05 were
considered statistically significant.