Participants
In 2019, we selected noise-exposed workers who had undergone occupational health checks from a number of automobile manufacturers in Guangzhou as participants, and the study was conducted from March to October in China. The selected research subjects have relatively fixed job positions and are less mobile in the production process. The study was approved by the Ethics Committee of the 12th People's Hospital of Guangzhou, and all subjects had provided informed consent. The study included 191 NIHL workers and 191 hearing-normal workers, and no workers were exposed to other occupational hazards. We selected 191 cases of noise exposed workers with binaural high-frequency hearing thresholds greater than 25dB (A). This control group was matched according to the following criteria: (1) same enterprises, types of work and operating positions as the case group; (2) binaural arbitrary frequency bands (including 500, 1000, 2000, 3000, 4000, 6000 Hz) hearing thresholds less than or equal to 25 dB (A); (3) same age (±3 years), same noise exposure time (±1 year).
The inclusion criteria for the subjects were as follows:
Cumulative time of occupational noise exposure [noise exposure time ≥ 8 h/day or 40 h/week, noise intensity ≥ 80 dB (A)] >1 year; (2) male and Han; (3) age: 18 ~ 45 year old.
The exclusion criteria were as follows:
Exposure to explosives or head injuries within 1 month prior to physical examination; (2) family history of hearing loss; (3) otitis or other otological diseases; (4) fever or common infections (flu, diarrhea and hepatitis,etc); (5) history of taking ototoxic drugs; (6) participants with bone conduction audiometry suggestive of conductive deafness.
The physical examination was performed by occupational health examiners in accordance with the standard protocol for each participant. Height, weight, blood pressure, blood lipids, pure tone audiometry were measured and we also inquired about the contact situation of other occupational hazards.We used EDTA anticoagulant negative pressure glass tubes to collect peripheral whole blood of empty-stomach subjects . The blood collection tube has a test tube number that can be one-to-one corresponding to the physical examination number, which ensures the consistency of the blood sample, the physical examination result and the questionnaire. Blood samples were temporarily stored and safely transported in a mobile refrigerator after collected. Questionnaires were collected by professionally trained investigators conducting face-to-face surveys and inquiries. The items of the questionnaire include general information, professional history, personal history, past history, personal conscious symptoms and so on.
On the basis of the "Diagnosis of Occupational Noise Deafness" (GBZ 49-2014) and relevant regulations and standards, specialized occupational health doctors performed at least 3 pure-tone hearing tests (the pure-tone hearing threshold test is performed in accordance with GB / T7583 and GB / T16403). Hearing thresholds for both ears were determined in increments of 5 dB in 500 dB, 1000 Hz, 2000 Hz, 3000 Hz, 4000 Hz, and 6000 Hz frequencies. According to GB / T7582–2004, the results were modified by age and gender. The PTA defines the hearing threshold at high frequencies as the average of each ear at 3000, 4000, and 6000 Hz. The hearing threshold at speech frequencies is defined as the average of 500, 1000, and 2000 Hz per ear. All subjects were required to avoid noise exposure for more than 48 hours before conducting audiometry.
The study conducted noise detection in accordance with "Measurement of physical factors in the workplace-Part 8: Noise" (GBZ / T189.8 - 2007). We measured noise after elaborative observation and investigation on the the working environment of spatial distribution, processing procedure, and noise equipment layout in these factories. We used the EDGE individual noise dosimeter produced by the British company CASELLA to evaluate the noise intensity. In this study, Noise exposure was evaluated with A-weighted energy equivalent continuous sound pressure level (Lex.8 h) according to the National Criteria of Measurement of Noise in the Workplace (GBZ/T189.8-2007) (China, 2007). Cumulative noise exposure (CNE) was calculated as
CNE = Lex.8 h + 10logT (Formula 2), where T means years of noise exposure.
SNP selection and genotyping
1. Screening of candidate gene SNP
Find the gene name in NCBI-SNP (http://www.ncbi.nlm.nih.gov/snp/). Open Gene View, refresh after clicking Clinical Source and in gene region. Select functional SNP sites of Promoter proxy (upstream variant 2KB), 5'UTR, Exon (missense, synonymous), 3'UTR region(Relevant optimization parameters are MAF in CHB> 0.05, based on HapMap or 1000 Genomes database) in this gene.
Mark disease-susceptible results by refering to relevant literature
Predict the function of the screened SNP (http://snpinfo.niehs.nih.gov/).
Perform LD analysis of the above SNP and mark the full linkage site of R2 = 1.
(http://asia.ensembl.org/Homo_sapiens/Tools/LD?db=core)
2. The SNP inclusion criteria are as follows:
Functional SNP sites located in Promoter proxy (upstream variant 2KB), 5'UTR, Exon (missense, synonymous), 3'UTR; (2) MAF in CHB> 0.05; (3) The linkage disequilibrium value of r2 is> 0.80; (4) Genetic balance test (Hardy-Weinberg) P value> 0.05. In this study, 14 SNP were screened for the three candidate genes TRIOBP, CASP3, and CASP7 (Table S1).
Table S1. Basic information, primer and probe sequences of selected SNP.
SNP
|
allele
|
MAF IN CHB
|
Function Prediction
|
Region
|
Primers for PCR
|
Probes for detecting variations
|
PHWE
|
CASP3
|
|
|
|
|
|
|
|
rs6948
|
G/T
|
0.199
|
--
|
3' UTR
|
F:GGAGGCCTCCCGGGCTGAG
|
TG:GGAGGCCTCCCGGGCTGAGG
TT:GGAGGCCTCCCGGGCTGAGT
|
0.107
|
rs1049216
|
G/A
|
0.209
|
miRNA binding site
|
3' UTR
|
R:TGAAAAAGTTAAACATTGAAGTAA
|
TG:TGAAAAAGTTAAACATTGAAGTAAC
TA:TGAAAAAGTTAAACATTGAAGTAAT
|
0.107
|
rs113420705
|
C/T
|
0.32
|
--
|
5' UTR
|
F:AGCCTCCTCATACCTTC
|
TC:AGCCTCCTCATACCTTCC
TT:AGCCTCCTCATACCTTCT
|
0.400
|
rs12108497
|
T/C
|
0.282
|
TFBS
|
promoter
|
R:GGACTCTGTGACTATAAAAGATG
|
TT:GGACTCTGTGACTATAAAAGATGA
TC:GGACTCTGTGACTATAAAAGATGG
|
0.607
|
rs1405937
|
C/G
|
0.277
|
TFBS
|
promoter
|
F:CCCCCAGGGACCCCATGGCA
|
TC:CCCCCAGGGACCCCATGGCAC
TG:CCCCCAGGGACCCCATGGCAG
|
0.848
|
rs4647602
|
G/T
|
0.288
|
TFBS
|
promoter
|
F:CTGCAGGGCCGAAAA
|
TG:CTGCAGGGCCGAAAAG
TT:CTGCAGGGCCGAAAAT
|
0.914
|
CASP7
|
|
|
|
|
|
|
|
rs2227310
|
C/G
|
0.427
|
nsSNP
|
Exon-missense
|
F:AGGGAGCACGGAAAAGA
|
TC:AGGGAGCACGGAAAAGAC
TG:AGGGAGCACGGAAAAGAG
|
0.610
|
rs12415607
|
C/A
|
0.417
|
TFBS
|
promoter
|
R:TTGAGTACATGCTTAGTGGTC
|
TC:TTGAGTACATGCTTAGTGGTCG
TA:TTGAGTACATGCTTAGTGGTCT
|
0.491
|
rs11196418
|
G/A
|
0.117
|
TFBS
|
promoter
|
R:CCCAAACACACAGATTCTAGTT
|
TG:CCCAAACACACAGATTCTAGTTC
TA:CCCAAACACACAGATTCTAGTTT
|
0.598
|
rs4353229
|
C/T
|
0.427
|
miRNA binding site
|
3' UTR
|
F:ACATGCAACAGAAGTGAC
|
TC:ACATGCAACAGAAGTGACC
TT:ACATGCAACAGAAGTGACT
|
0.611
|
rs10787498
|
G/T
|
0.194
|
miRNA binding site
|
3' UTR
|
F:CAGTGGTAGAGTCATGT
|
TG:CAGTGGTAGAGTCATGTG
TT:CAGTGGTAGAGTCATGTT
|
0.251
|
rs12247479
|
G/A
|
0.112
|
miRNA binding site
|
3' UTR
|
R:CCATTGGTGGTCCTAA
|
TG:CCATTGGTGGTCCTAAC
TA:CCATTGGTGGTCCTAAT
|
0.129
|
rs1127687
|
A/G
|
0.204
|
miRNA binding site
|
3' UTR
|
F:CAGCCATGACAAGAACAAA
|
TA:CAGCCATGACAAGAACAAAA
TG:CAGCCATGACAAGAACAAAG
|
0.881
|
rs12263370
|
A/G
|
0.141
|
TFBS
|
promoter
|
F:GGAAGTAAGCCACCGCGCCT
|
TA:GGAAGTAAGCCACCGCGCCTA
TG:GGAAGTAAGCCACCGCGCCTG
|
0.193
|
Primer design and dilution
Sort the rs numbers of the sites before detection, and input the rs numbers to http://agenacx.com/ for primer design. According to the results of the operation, select and determine the appropriate primer design scheme, and order primers. The PCR primer was diluted to 100 μM, and a PCR primer mixture was prepared according to 1: 200. The extension primers were diluted according to the dilution table. Prepare the EXT primer mix at 1:25. After the extension primer mixture was prepared, 2 μl was diluted 25-fold for mass spectrometry. Adjust the extension primer ratio of individual sites according to the test results.
DNA extraction
Sample DNA was extracted using a ThermoFisher automated magnetic bead extractor. For blood samples, the Magpure Buffy Coat DNA Midi KF Kit kit was used. The NanoDrop8000 instrument was used for OD value detection and 1.25% agarose gel electrophoresis. After passing the DNA quality test, the sample DNA were transferred to a 96-well plate and stored at -20 ° C.
Agena MassArray System Genotyping Steps
A target fragment containing the SNP site to be detected was amplified by a PCR reaction. Shrimp alkaline phosphatase (SAP enzyme) was then used to remove the remaining deoxyribonucleoside triphosphate (dNTP) and primers in the PCR system. Then single base extension primers was added, the 3 'terminal base of which is close to the SNP site and is completely complementary to the base on the target fragment. Four types of ddNTP were used instead of dNTP. The probe extended only one base at the SNP site, and the ddNTP on the connection corresponds to the allele of the SNP site. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to detect the molecular weight difference between the extended product and the unextended primer, and the base at this point was determined.
The PCR master mix was configured and oscillated at low speed. We added 4 μl PCR master mix to each well of the 384-well plate, and mixed them after 1 μl of template DNA (20ng / μl) was added. The PCR reaction plate was placed on the PCR instrument and then the program was started. After the PCR reaction was completed, the PCR products were treated with SAP to remove free dNTPs from the system. Next, we prepared alkaline phosphatase treatment in a new 1.5ml EP tube, following by adding the SAP mix to a 384-well PCR reaction plate. After centrifugation, the SAP reaction program was performed, and then a single base extension reaction was activated after the completion of alkaline phosphatase treatment. Subsequently, we prepared a single base extension reaction solution in a new 1.5ml EP tube, and added the EXTEND Mix to the 384-well reaction plate. Again with centrifugation, an extension reaction procedure was performed. The cation exchange resin was used to remove Na +, Mg2 +, K + and other salt ions after the PCR reaction, so as to avoid excessive salt peaks in the analysis spectrum produced by mass detection, which would affect the result judgment. The PCR product plate was contrifuged for 5min (4000r / min), and 19ul of ultrapure water was added to each reaction well and centrifuged for 1min. Resin was applied on the top, and the PCR product plate was left to dry at room temperature for 15-30 minutes. Afterwards, they were mixed up for 40 minute to 1 hour. The sample was then micro-loaded onto a SpectroCHIP with a Mass Array Nanodispenser to prepare a co-crystallized film of the chip matrix and the sample. The prepared chip was put into a mass spectrometer (MassARRAY Analyzer 4 System) for detection, and Typer 4.0 software was used to obtain the original data and cluster map, and check the integrity and accuracy of the data file.`
Statistical Analysis
Categorical variables were expressed in frequency (%) and analyzed by Pearson's χ2 independence test. Quantitative variables obeying normal distribution were expressed as mean ± standard deviation (M ± SD) and analyzed by Student's t test. We performed a χ2 goodness-of-fit test on the frequency of each genotype tested to verify that it complies with Hardy-Weinberg Equilibrium (HWE). Conditional logistic regression was used to correct the statistically significant confounding factors in the study. The OR (Odds Ratio) value and its 95% CI (Confidence interval) were used to analyze the correlation between the gene SNP and the risk of NIHL. We used stratified and crossover analysis methods to analyze the potential relationship between genes and the environment. Multifactor dimensionality reduction (MDR) method was used to explore the potential interactions between genes. Statistical analysis was performed using SAS version 9.2 (SAS INSTITUTE INC, Cary, NCSU, USA). The MDR method uses MDR version 3.0.2 (Computational Genetics Laboratory of the University of Pennsylvania, Philadelphia, PA, USA). We considered all significant statistical tests with p value<0.05 . Bonferroni correction was applied to multiple hypothesis tests.