Subjects
This study included 134 newly diagnosed patients with clinically diagnosed non-Hodgkin lymphoma and 125 healthy controls with no history of cancer or inflammatory diseases were recruited for the study. patients were histologically confirmed as having non-Hodgkin lymphoma at the Ali Ebn Abitaleb Hospital of Zahedan University(Sistan and Baluchestan Province.Iran). 284 subjects were randomly selected from individuals who were participating in a routine cancer-screening program for the early detection of non-Hodgkin lymphoma cancer during the same period. All the control subjects were found to lack non-Hodgkin lymphoma lesions by cytology test. Two controls were matched to each case by age at enrollment (within ±5 years).
Selection criteria
We evaluated studies on patients with non-Hodgkin lymphoma cancer and PD1/PDL1 genetic polymorphisms as risk factors. The following inclusion criteria were applied to assess each publication for inclusion: (1) independent case-control study that evaluated the relationship between PD1/PDL1 genetic polymorphisms and the risk of non-Hodgkin lymphoma cancer; (2) all patients diagnosed with non-Hodgkin lymphoma cancer were confirmed by histopathological examinations demonstrating the occurrence of invasion; (3) the number of evaluated cancer cases was provided; (4) at least 134 cases were included in the study; (5) the genotype number and frequency information were supplied. The exclusion criteria were the following: (1) studies on familial and hereditary non-Hodgkin lymphoma cancer; and (2) studies on haplotypes alone. If the same population was included in previous studies, only the most recent or largest sample size study was included.
Ethical statements
All patients and controls signed written informed consent before participating, and the protocol of this study was approved by the institutional Ethnics Committee of Ali Ebn Abitaleb Hospital of Zahedan University(Sistan and Baluchestan Province.Iran).
Sample collection
Ten mL of venous blood were extracted from all subjects after fasting for more than 12 h. The blood samples (4 mL) were anticoagulated with ethylenediaminetetraacetic acid (EDTA) and stored at −70°C. Then the samples were incubated in an upright position for 1 h, followed by centrifuging at 3000 rpm for 10 min at room temperature to isolate the peripheral blood mononuclear cells. Afterwards, the genomic DNA was isolated using a DNA extraction kit (Cat no.: DP318-03, Tiangen Biotech Beijing Co. Ltd., Beijing, China) according to the manufacturer’s instructions. The remaining 6 mL blood samples were incubated in an upright position for 1 h and were centrifuged at 3000 rpm for 10 min at room temperature. Subsequently, serum were extracted and stored at −70°C until used.
SNP detection
Four SNP sites, PD-L1 rs2890658 C>A, PD-1 rs2227981 C>T , PD-1 rs11568821 G>A and PD-L1 rs4143815 G>C were selected to conduct the current research. Locations and base pair positions of single nucleotide polymorphisms (SNPs) in PD-1 and PD-L1 genes are present in Table1. Restriction fragment length polymorphism polymerase chain reaction (PCR-RFLP) was used to analyze the genotype and allele frequency in 4 sites between the case and control groups. The PCR primers were designed using Primer Premier 5.0 software and synthesized by Shanghai Chemical Company (China). The amplification sites, primer sequences, fragment length, annealing temperature, and cycle number for the PCR-RFLP are presented in Table 2. The total volume of PCR reaction included 20 μl: 2 μl of 10×PCR reaction buffer, 2 μl of deoxy-ribonucleoside triphosphate (dNTP) (2.5 mmol/L for each), 0.5 μl of each forward and reverse primer (10 pmol/μl), 2.5 U of Platinum Taq DNA polymerase (Invitrogen, Shanghai, China), and 50 ng of genomic DNA. DNA was amplified during 30 cycles with 5 min predegeneration at 95°C, 30 s denaturation at 95°C, 30 s annealing at 58°C, and 40 s extension at 72°C. Then the DNA was further extended for 5 min at 72°C and stored at 4°C. A total of 20 μl PCR production was extracted to construct the enzyme reaction system. Then the PCR products were digested with restricted enzyme RsaI and XmnI (New England Biolabs LTD., Beijing). After incubation at 21°C overnight, the PCR products were placed in a water bath at 4°C for 15 min to terminate the reaction. Then the enzyme-digested products were electrophoresis-separated by 2% agarose gel (containing ethidium bromide). The gel imaging system (Bio-Rad, USA) was used for genotyping interpretation. . The primers used for detection of PD-1 and PD-L1 polymorphisms using PCR-RFLP are presented in Table3.
Table 1. Locations and base pair positions of single nucleotide polymorphisms (SNPs) in PD-1 and PD-L1 genes.
Gene Name
|
db SNP rs # ID a
|
Chromosome Position
|
Location
|
Base Change
|
Amino Acid Change
|
PD-1
|
rs2227981
|
chr2:241851121
|
Upstream
|
C/T
|
-
|
|
rs11568821
|
chr2:241851760
|
Intron
|
G/A
|
-
|
PD-L1
|
rs4143815
|
chr9:5468257
|
3′UTR
|
G/C
|
-
|
|
rs2890658
|
chr9:5465130
|
Intron
|
A/C
|
|
Table 2 The PCR primer sequences for PD1/PDL1 single nucleotide polymorphisms.
Gene
|
SNP
|
Primer sequences
|
Annealing temperature (°C)
|
Circles
|
PD-1
|
rs2227981 C>T
|
F: TGAGCAGACGGAGTATGCC
R: CTGAGGAAATGCGCTGACC
|
59
|
30
|
PD-1
|
rs11568821
G>A
|
F: CTCACATTCTATTATAGCCAGGACC
R: TAAGATAAGAAATGACCAAGCCCAC
|
68
|
30
|
PD-L1
|
rs4143815 G>C
|
F: 5′-CCCCCATCATGTCTCCTCTCC-3′ R: 5′-CCAAGCAACTTGGTGTTTTGAGG-3′
|
67
|
30
|
PD-L1
|
rs2890658 C>A
|
F: GCAAGAGGAAGTGAAATAATCAAG
R: GATACCTGTGTTAAAATGGGAACAG
|
61
|
30
|
Table 3. The primers used for detection of PD-1 and PD-L1 polymorphisms using PCR-RFLP
polymorphism
|
Restriction Enzyme
|
Fragment (bp)
|
PD-L1 rs2890658 C>A
|
HaeIII
|
CC: 226+25
CA: 251+226+25
AA: 251
|
PD-1 rs2227981 C>T
|
PvuII,
|
CC: 207
TC: 207+133+74
TT: 133+74
|
PD-1 rs11568821
G>A
|
PstI
|
GG: 290
AG:290+197+93
AA: 197+93
|
PD-L1 rs4143815 G>C
|
Hpy8I
|
CC: 227
CG: 227+199+28
GG: 199+28
|
Viral RNA extraction and molecular detection
Viral RNA was extracted from 200 μl fluid samples by High Pure Viral Nucleic Acid kit (Roche, Germany) according to the manufacturer’s manual. Then, Real-time RT-PCR was performed by RT-PCR kit (QIAGEN, Germany) using specific primers and probes in Stepone pulse instruments (Applied Biosystems, Foster City, CA) according to the protocol. The primer sequences used to identify are listed in Table 4.
Table 4 Real-time PCR primers and probes specifications
|
Forward primer
|
Revers primer
|
Product Length
|
Sequence (5'->3')
|
GC%
|
length
|
Sequence (5'->3')
|
GC%
|
length
|
PD-1
|
CCGCACGAGGGACAATAG
|
61.1
|
18
|
GGTGGCATACTCCGTCTG
|
61.1
|
18
|
167
|
PD-L1
|
AGGGCATTCCAGAAAGATGAGG
|
50
|
22
|
GGGAACCGTGACAGTAAATGCG
|
54.55
|
22
|
88
|
Statistical analysis
Statistical analysis was conducted using SPSS 18.0 software (IBM Corporation, Somers, NY, USA). Continuous data are expressed as χ̄ ± standard deviation (SD), using t test or variance analysis for comparisons. Categorical data was presented with percentages and chi-square test was applied for comparisons between groups. Chi-square test was also used to verify whether the genotype distribution of the 4 SNPs met Hardy-Weinberg (HW) equilibrium. The genotype frequency and allele frequency between the case and control were calculated by OR (odds ratio) with 95%CI (confidence interval). All tests were 2-sided and differences were considered statistically significant at P<0.05.The association between PD-L1 rs2890658 C>A, PD-1 rs2227981 C>T , PD-1 rs11568821 G>A and PD-L1 rs4143815 G>C PD1/PDL1 SNPs and non-Hodgkin lymphoma was assessed using OR with 95%CI.