**3. Results**

#### **3.1 The characteristics of subjects**

All subjects suffered from hepatic carcinoma, and **Table 1** summarized their characteristics. The mean age of all participants was 50.74 11.55 years, and more than 70% of them are male. For these cancer patients, 70.3% (267/380) and 26.3% (100/380) cases featured TNM II and III stages of tumor, and they also had an average AFB1 exposure value of 2.27 1.09 μmol/mol DNA.

#### **3.2 XRCC4 GSNPs increased AFB1-DNA adducts**

A total of 143 GSNPs in the coding regions of XRCC4 gene were selected in our final analyses, and **Table 2** showed the genotypic distribution of all GSNPs. To evaluate the effects of these potential GSNPs on AFB1-DNA adducts, the role of each GSNP in the coding regions of XRCC4 gene was tested using Student *t-*test or ANOVA test with the adjustment of multiple test. Among these GSNPs, only rs1237462915 (cat#SNP016, at codon 38), rs28383151 (cat#SNP026, at codon 56), rs762419679 (cat#SNP069, at codon 127), rs766287987 (cat#SNP112, at codon 203),


#### **Table 1.**

*The characteristics of subjects.*

and rs3734091 (cat#SNP138, at codon 247) significantly affected the levels of AFB1-DNA adducts in the tumor tissues with HCC. The adduct amounts of their wild genotypes (defined as XX genotype) were 2.15 0.97 μmol/mol DNA, 2.07 0.99 μmol/mol DNA, 2.12 0.86 μmol/mol DNA, 2.11 0.89 μmol/mol DNA, and 2.09 0.97 μmol/mol DNA, respectively. For their mutant heterozygotic genotypes (defined as XY genotype), the amounts of AFB-DNA adduct were from 2.64 to 4.33 μmol/mol DNA, whereas the adduct levels were from 3.04 to 5.78 for the mutant homozygotic genotypes (defined as YY genotype) (**Table 2**).

Additionally, mutant genotypes of several other GSNPs, including rs761695470 (SNP008, at codon 18), rs758779099 (SNP018, at codon 40), rs144653114 (SNP054, at codon 103), rs1277864722 (SNP085, at codon 153), and rs777195630 (SNP100, at codon 180), also increased the amounts of AFB1-DNA adducts; however, they had no statistical significance according to screening threshold value.

#### **3.3 XRCC4 GSNPs modified the AFB1-related HCC prognosis**

Because the poor prognosis of patients with HCC has been associated with the toxicity of AFB1, we followed up the survival information of all patients and explored whether positive GSNPs of XRCC4 modified HCC outcomes, including overall survival (OS) and disease recurrence-free survival (RFS) (**Figures 1** and **2**). Results from Kaplan-Meier survival model (based on the cumulative risk models) and Cox regression model analyses showed that compared with their wild

**No.**

**157**

SNP001

SNP002

SNP003

SNP004

SNP005

SNP006

SNP007

SNP008

SNP009

SNP010

SNP011

SNP012

SNP013

SNP014

SNP015 SNP016

SNP017

SNP018

 rs758779099

 5:83105039

 T/G

rs748540743

 5:83105032

 A/T

AA/AT/TT TT/TG/GG

38 40

H/Q

 353/25/2

 2.23

 2.63

 0.01

1.03/2.78

 1.61/

0.04

D/V

 380/0/0

rs1237462915

 5:83105031

 G/T

rs1232077487

 5:83105022

 A/G

rs1305679408

 5:83105013

 T/A

rs1484250582

 5:83105006

 A/C

 rs550773308

 5:83104999

 C/T

rs1288681786

 5:83104992

 G/A

GG/GA/AA CC/CT/TT AA/AC/CC TT/TA/AA AA/AG/GG GG/GT/TT

25 27 29 32 35 38

D/Y

 352/19/9

 2.15

0.97/3.62

4.21

2.27

 1.09/

/

—

 0.93

 1.40/

2.96 105

T/A

 380/0/0

F/I

 350/30/0

 2.27

 2.27

 1.09/

/

—

1.08/2.26

 1.14/

0.96

E/D

 376/4/0

 2.27

1.09/2.18

 1.42/

0.87

T/I

 367/13/0

 2.26

1.06/2.51

 1.80/

0.63

E/K

 354/26/0

 2.28

1.10/2.16

 0.96/

0.56

rs1271180927

 5:83104986

 T/A

 rs755776572

 5:83104983

 G/A

GG/GA/AA TT/TA/AA

22 23

S/T

 364/13/3

 2.26

1.09/2.52

2.44

 0.77

 1.04/

0.89

V/I

 380/0/0

 rs761695470

 5:83104971

 C/T

 rs774071130

 5:83104966

 T/C

TT/TC/CC CC/CT/TT

16 18

H/Y

 371/6/3

 2.24

1.03/2.87

4.42

2.27

 1.09/

/

—

 2.93

 2.00/

0.04

*X-Ray Repair Cross Complementing 4 (XRCC4) Genetic Single Nucleotide Polymorphisms…*

I/T

 370/10/0

 2.28

1.09/2.05

 0.94/

0.99

 rs28383138

 5:83104954

 C/G

rs1252823908

 5:83104946

 C/A

rs1425642930

 5:83104933

 T/G

rs1166890864

 5:83104930

 A/G

rs1449631425

 5:83104929

 A/G

AA/AG/GG AA/AG/GG TT/TG/GG CC/CA/AA CC//CG/GG

4 4 5 9 12

S/C

 319/47/14

 2.26

1.09/2.41

2.00

 1.07

 1.06/

0.23

H/Q

 376/4/0

 2.27

1.09/2.16

 0.56/

0.94

I/R

 376/4/0

 2.27

1.09/2.10

 0.61/

0.96

*DOI: http://dx.doi.org/10.5772/intechopen.88752*

K/R

 378/2/0

 2.27

1.09/1.57

 0.03/

0.99

K/E

 377/3/0

 2.27

1.09/2.21

 0.54/

0.99

 rs142575170

 5:83104923

 G/A

 **SNP ID**

 **Chr: bp**

 **Alleles**

**Genotypes**

**Amino acid**

**Amino acid**

**Nxx/xy/yyc**

**Adducts (mean**

 **SD)**

*P***d**

**change**

**position**

2

E/K

 323/54/3

 2.28

 2.36

 1.05

1.09/2.21

 1.08/

0.23

**(xx/xy/yy)b**

GG/GA/AA


#### *X-Ray Repair Cross Complementing 4 (XRCC4) Genetic Single Nucleotide Polymorphisms… DOI: http://dx.doi.org/10.5772/intechopen.88752*

and rs3734091 (cat#SNP138, at codon 247) significantly affected the levels of AFB1-DNA adducts in the tumor tissues with HCC. The adduct amounts of their wild genotypes (defined as XX genotype) were 2.15 0.97 μmol/mol DNA, 2.07 0.99 μmol/mol DNA, 2.12 0.86 μmol/mol DNA, 2.11 0.89 μmol/mol DNA, and 2.09 0.97 μmol/mol DNA, respectively. For their mutant heterozygotic genotypes (defined as XY genotype), the amounts of AFB-DNA adduct were from 2.64 to 4.33 μmol/mol DNA, whereas the adduct levels were from 3.04 to 5.78 for

Total 380 100.0

*Aflatoxin B1 Occurrence, Detection and Toxicological Effects*

≤35 53 13.9 –40 38 10.0 –45 55 14.5 –50 43 11.3 –55 56 14.7 –60 42 11.1 –65 49 12.9 ≥66 44 11.6

Male 271 71.3 Female 109 28.7

Han 221 58.2 Zhuang 159 41.8

I–II 13 3.4 III 267 70.3 IV 100 26.3

Age (years)

Gender

Race

**Table 1.**

**156**

TNM stage

*The characteristics of subjects.*

**n %**

the mutant homozygotic genotypes (defined as YY genotype) (**Table 2**).

(SNP008, at codon 18), rs758779099 (SNP018, at codon 40), rs144653114 (SNP054, at codon 103), rs1277864722 (SNP085, at codon 153), and rs777195630 (SNP100, at codon 180), also increased the amounts of AFB1-DNA adducts; however, they had no statistical significance according to screening threshold value.

**3.3 XRCC4 GSNPs modified the AFB1-related HCC prognosis**

Additionally, mutant genotypes of several other GSNPs, including rs761695470

Because the poor prognosis of patients with HCC has been associated with the

toxicity of AFB1, we followed up the survival information of all patients and explored whether positive GSNPs of XRCC4 modified HCC outcomes, including overall survival (OS) and disease recurrence-free survival (RFS) (**Figures 1** and **2**). Results from Kaplan-Meier survival model (based on the cumulative risk models)

and Cox regression model analyses showed that compared with their wild


**No.**

**159**

SNP039

SNP040

SNP041

SNP042

SNP043

SNP044

SNP045

SNP046

SNP047

SNP048

SNP049

SNP050

SNP051

SNP052

SNP053

SNP054 SNP055

SNP056

SNP057

SNP058

rs1240771489

 5:83195795

 A/G

rs1378785946

 5:83195794

 G/C

 rs757113391

 5:83195787

 C/G

CC//CG/GG GG/GC/CC AA/AG/GG

111 114 114

E/G

 281/80/19

 2.297

1.09/2.197

 1.06/

0.75

E/Q

 360/20/0

 2.277

1.10/2.227

 0.84/

0.83

F/L

 380/0/0

 rs79561451

 5:83195782

 T/C

TT/TC/CC

110

S/P

 352/25/3

 2.28

1.11/2.20

1.82

2.27

 1.09/

/

—

 0.37

 0.84/

0.74

rs144653114

 5:83111195

 G/A

rs1472843465

 5:83111180

 G/A

rs1236326383

 5:83111177

 T/C

rs1184763400

 5:83111174

 T/A

rs1239084198

 5:83111171

 T/G

 rs772190420

 5:83111169

 A/G

rs1049631686

 5:83111162

 T/A

 rs149355996

 5:83111147

 A/T

 rs756247552

 5:83111142

 C/T

rs1398927737

 5:83111141

 A/C

 rs572613361

 5:83111138

 T/C

rs757644947

 5:83111126

 C/T

rs528259464

 5:83111121

 C/T

 rs764572419

 5:83111120

 G/T

 rs763501478

 5:83111117

 G/A

 rs61762970

 5:83111112

 T/C

 **SNP ID**

 **Chr: bp**

 **Alleles**

**Genotypes**

**Amino acid**

**Amino acid**

**Nxx/xy/yyc**

**Adducts (mean**

 **SD)**

*P***d**

**change**

**position**

75 77 78 78 80 84 85 85 87 92 94 95 96 97 98 103

N/D

 240/127/

13

E/K

 377/3/0

 2.27 2.17

1.09/2.48

2.11

 0.84

 1.08/

0.03

1.09/2.58

 0.84/

0.63

F/L

 366/14/0

 2.28

1.09/1.92

 0.92/

0.24

F/I

 380/0/0

F/V

 356/24/0

 2.26

 2.27

 1.09/

/

—

1.07/2.36

 1.34/

0.68

Y/C

 354/26/0

 2.28

1.08/2.08

 1.23/

0.36

S/T

 364/26/0

N/Y

 371/9/0

 2.27

 2.27

 1.09/

/

—

*X-Ray Repair Cross Complementing 4 (XRCC4) Genetic Single Nucleotide Polymorphisms…*

1.10/2.36

 0.70/

0.81

T/M

 374/6/0

 2.26

1.08/2.56

 1.35/

0.50

T/P

 380/0/0

2.27

 1.09/

/

—

Y/H

 351/27/2

 2.27

 /2.24

1.02/3.09

 0.47

 0.57

P/S

 369/11/0

 2.26

1.07/2.51

 1.51/

0.46

*DOI: http://dx.doi.org/10.5772/intechopen.88752*

A/V

 376/4/0

 2.26

1.09/2.77

 0.44/

0.36

A/S

 349/31/0

 2.26

1.09/2.39

 1.09/

0.52

G/R

 361/29/0

L/S

 356/24/0

 2.29

 2.27

 1.09/

/

—

1.10/1.99

 0.89/

0.20

**(xx/xy/yy)b**

TT/TC/CC GG/GA/AA GG/GT/TT CC/CT/TT CC/CT/TT TT/TC/CC AA/AC/CC CC/CT/TT AA/AT/TT TT/TA/AA AA/AG/GG TT/TG/GG TT/TA/AA TT/TC/CC GG/GA/AA GG/GA/AA


#### *X-Ray Repair Cross Complementing 4 (XRCC4) Genetic Single Nucleotide Polymorphisms… DOI: http://dx.doi.org/10.5772/intechopen.88752*

**No.**

**158**

SNP019

SNP020

SNP021

SNP022

SNP023

SNP024

SNP025

SNP026

SNP027

SNP028

SNP029

SNP030

SNP031

SNP032

SNP033

SNP034

SNP035

SNP036

SNP037

SNP038

 rs371307071

 5:83111100

 G/C

 rs759064378

 5:83111097

 T/C

rs1478505961

 5:83111093

 G/A

rs1423928075

 5:83111090

 G/A

 rs776362814

 5:83111087

 G/A

rs1248297453

 5:83111085

 A/G

rs1285557699

 5:83111073

 A/G

 rs867574505

 5:83111072

 G/A

rs1369565470

 5:83111071

 G/C

rs760546308

 5:83111070

 T/C

rs748124509

 5:83111066

 G/T

rs1245127767

 5:83111063

 A/G

AA/AG/GG GG/GT/TT TT/TC/CC GG/GC/CC GG/GA/AA AA/AG/GG AA/AG/GG GG/GA/AA GG/GA/AA GG/GA/AA

TT/TC/CC GG/GC/CC

59 60 61 61 62 62 66 67 68 69 70 71

R/T

 380/0/0

L/P

 354/26/0

 2.28

 2.27

 1.09/

/

—

1.09/2.15

 1.01/

0.58

E/K

 250/28/2

 2.27

1.07/2.24

1.28/2.69

 1.16

 0.86

G/S

 380/0/0

V/I

 350/30/0

 2.26

 2.27

 1.09/

/

—

1.11/2.31

 0.84/

0.83

Y/C

 351/29/0

 2.26

1.07/2.36

 1.25/

0.63

E/G

 380/0/0

E/K

 380/0/0

M/I

 380/0/0

M/T

 352/28/0

 2.27

 2.27

2.27

2.27

 1.09/

/

—

 1.09/

/

—

 1.09/

/

—

1.04/2.25

 1.56/

0.91

A/S

 354/23/3

 2.25

1.11/2.36

0.74/2.78

 1.01

 0.66

M/V

 354/25/1

 2.27

1.10/2.18

0.92/2.78\*

0.67\*

 rs28383151

 5:83111054

 G/A

 rs945155269

 5:83111049

 A/C

rs868207005

 5:83111039

 G/A

rs1326359694

 5:83111033

 G/A

rs1248835327

 5:83111031

 C/T

rs1460004120

 5:83105053

 G/C

GG/GC/CC CC/CT/TT GG/GA/AA GG/GA/AA AA/AC/CC GG/GA/AA

45 48 49 51 54 56

A/T

 277/63/40

 2.07

 3.04

 0.78

0.99/2.64

 1.33/

2.83 1055

Q/P

 364/26/0

 2.29

1.07/2.02

 1.04/

0.24

E/K

 380/0/0

E/K

 355/25/0

 2.28

 2.27

 1.09/

/

—

1.11/2.16

 0.76/

0.61

*Aflatoxin B1 Occurrence, Detection and Toxicological Effects*

S/F

 370/10/0

 2.27

1.09/2.21

 1.01/

0.87

G/A

 375/5/0

 2.27

1.09/2.21

 0.82/

0.91

 rs771111816

 5:83105050

 C/T

 rs587779351

 5:83105046

 T/C

 **SNP ID**

 **Chr: bp**

 **Alleles**

**Genotypes**

**Amino acid**

**Amino acid**

**Nxx/xy/yyc**

**Adducts (mean**

 **SD)**

*P***d**

**change**

**position**

43 44

T/I

 362/25/3

 2.26

 2.68

 0.07

1.08/2.33

 1.23/

0.78

W/R

 380/0/0

2.27

 1.09/

/

—

**(xx/xy/yy)b**

TT/TC/CC CC/CT/TT


**No.**

**161**

SNP079

SNP080

SNP081

SNP082

SNP083

SNP084

SNP085

SNP086

SNP087

SNP088

SNP089

SNP090

SNP091

SNP092

SNP093

SNP094

SNP095

SNP096

SNP097

SNP098

SNP099

 rs771544881

 5:83203605

 G/A

rs140143447

 5:83203604

 C/T

 rs777300742

 5:83203599

 A/G

 rs778422015

 5:83203595

 C/T

rs1376295451

 5:83203587

 A/T

 rs753605351

 5:83203575

 A/C

rs1359488275

 5:83203568

 A/T

 rs375731584

 5:83203566

 T/G

rs1325151692

 5:83203554

 T/A

rs1156689163

 5:83203553

 T/G

 rs372493882

 5:83195933

 G/T

rs369499884

 5:83195930

 A/G

rs1201811742

 5:83195924

 A/C

 rs745751715

 5:83195922

 T/G

rs1277864722

 5:83195912

 G/A

rs1375107569

 5:83195905

 C/T

 rs757278630

 5:83195903

 G/A

rs1022211508

 5:83195897

 A/G

 rs553111266

 5:83195883

 C/A

CC/CA/AA AA/AG/GG GG/GA/AA CC/CT/TT GG/GA/AA TT/TG/GG AA/AC/CC AA/AG/GG GG/GT/TT TT/TG/GG TT/TA/AA TT/TG/GG AA/AT/TT AA/AC/CC AA/AT/TT CC/CT/TT AA/AG/GG CC/CT/TT GG/GA/AA

143 148 150 151 153 156 157 159 160 162 162 166 167 169 173 176 177 179 179

R/Q

 380/0/0

R/W

 372/8/0

 2.28

 2.27

 1.09/

/

—

1.09/1.69

 0.56/

0.13

Y/C

 354/26/0

 2.26

1.08/2.39

 1.24/

0.57

L/F

 380/0/0

E/V

 340/40/0

 2.28

 2.27

 1.09/

/

—

1.10/2.18

 0.98/

0.58

K/T

 385/15/0

 2.26

1.10/2.40

 0.89/

0.64

S/C

 380/0/0

V/G

 355/25/0

 2.25

 2.27

 1.09/

/

—

1.09/2.48

 0.97/

0.32

F/Y

 345/95/0

 2.26

1.04/2.39

 1.46/

0.49

F/V

 380/0/0

G/V

 250/30/0

 2.29

 2.27

 1.09/

/

—

1.10/2.07

 0.97/

0.30

Q/R

 367/13/0

 2.28

1.09/2.03

 1.08/

0.42

D/A

 353/26/1

 2.24

1.07/2.67

1.22/2.72e

0.13

*X-Ray Repair Cross Complementing 4 (XRCC4) Genetic Single Nucleotide Polymorphisms…*

N/K

 289/89/2

 2.28

1.04/2.25

1.23/2.12

 1.83

 0.97

R/K

 355/25/0

 2.29

1.01/1.93

 0.72/

0.02

L/F

 380/0/0

R/K

 378/12/0

 2.27

 2.27

 1.09/

/

—

1.09/1.99

 1.01/

0.36

N/S

 342/38/0

 2.30

1.11/2.03

 0.79/

0.15

*DOI: http://dx.doi.org/10.5772/intechopen.88752*

H/Q

 373/7/0

 2.26

1.09/2.75

 1.07/

0.24

 rs28360136

 5:83195878

 G/C

 rs374892515

 5:83195876

 A/G

 **SNP ID**

 **Chr: bp**

 **Alleles**

**Genotypes**

**Amino acid**

**Amino acid**

**Nxx/xy/yyc**

**Adducts (mean**

 **SD)**

*P***d**

**change**

**position**

141 142

E/Q

 254/111/

2.33

1.10/2.12

1.02/2.35

 1.26

 0.21

15

N/S

 380/0/0

2.27

 1.09/

/

—

**(xx/xy/yy)b**

AA/AG/GG GG/GC/CC


#### *X-Ray Repair Cross Complementing 4 (XRCC4) Genetic Single Nucleotide Polymorphisms… DOI: http://dx.doi.org/10.5772/intechopen.88752*

**No.**

**160**

SNP059

SNP060

SNP061

SNP062

SNP063

SNP064

SNP065

SNP066

SNP067

SNP068

SNP069

SNP070

SNP071

SNP072

SNP073

SNP074

SNP075

SNP076

SNP077

SNP078

rs370037164

 5:83195869

 G/A

 rs56334522

 5:83195865

 T/G

rs1384832919

 5:83195863

 A/G

 rs372774793

 5:83195857

 G/C

rs1335801774

 5:83195856

 T/G

TT/TG/GG GG/GC/CC AA/AG/GG TT/TG/GG GG/GA/AA

134 135 137 137 139

A/T

 292/84/4

 2.28

 2.49

 0.83

1.06/2.23

 1.18/

0.89

N/K

 366/13/1

 2.26

1.10/2.53

0.80/1.34b

 0.89

N/D

 368/12/0

 2.27

1.10/2.00

 0.59/

0.38

A/P

 358/22/0

 2.27

1.06/2.25

 1.46/

0.92

I/M

 380/0/0

 rs28360135

 5:83195855

 T/C

rs1484047716

 5:83195852

 C/G

rs1178870682

 5:83195848

 G/C

rs1412506484

 5:83195840

 A/G

 rs762419679

 5:83195834

 T/C

rs1159852376

 5:83195824

 A/G

rs1198491910

 5:83195821

 A/G

rs1395194011

 5:83195819

 T/A

 rs369641536

 5:83195816

 A/G

 rs768175717

 5:83195810

 C/T

 rs375157105

 5:83195809

 C/A

 rs550178738

 5:83195807

 A/T

rs1301137729

 5:83195800

 G/C

rs1056939125

 5:83195798

 A/C

rs769556494

 5:83195797

 A/G

 **SNP ID**

 **Chr: bp**

 **Alleles**

**Genotypes**

**Amino acid**

**Amino acid**

**Nxx/xy/yyc**

**Adducts (mean**

 **SD)**

*P***d**

**change**

**position**

115 115 116 118 119 119 121 122 123 124 127 129 132 133 134

I/T

 304/64/12

 2.31

 2.12

2.27

 1.09/

/

—

 0.69

1.13/2.12

 0.90/

0.39

T/S

 374/6/0

 2.26

1.07/2.71

 1.90/

0.32

D/H

 365/15/0

 2.27

1.10/2.26

 0.78/

0.98

Y/C

 349/31/0

 2.27

1.10/2.26

 1.32/

0.96

I/T

 359/15/6

 2.12

0.86/4.33

1.99/5.99

 1.12 1.17 1043

R/G

 369/11/0

 2.26

1.10/2.40

 0.88/

0.64

I/V

 348/32/0

 2.27

1.09/2.25

 1.13/

0.95

V/D

 289/81/10

 2.24

1.07/2.62

 1.22/

0.05

E/G

 380/0/0

P/L

 380/0/0

P/T

 352/28/0

 2.29

 2.27

2.27

 1.09/

/

—

 1.09/

/

—

*Aflatoxin B1 Occurrence, Detection and Toxicological Effects*

1.102.06

 0.83/

0.28

N/I

 341/39/0

 2.27

1.10/2.28

 1.00/

0.93

V/L

 359/21/0

 2.27

1.09/2.19

 0.99/

0.74

K/T

 380/0/0

K/E

 340/40/0

 2.277

 2.27

 1.09/

/

—

1.09/2.277

 1.04/

0.99

**(xx/xy/yy)b**

AA/AG/GG AA/AC/CC GG/GC/CC AA/AT/TT CC/CA/AA CC/CT/TT AA/AG/GG TT/TA/AA AA/AG/GG AA/AG/GG

TT/TC/CC AA/AG/GG GG/GC/CC CC//CG/GG

TT/TC/CC


**No.**

**163**

SNP120

SNP121

SNP122

SNP123

SNP124

SNP125

SNP126

SNP127

SNP128

SNP129

SNP130

SNP131

SNP132

SNP133

SNP134 SNP135

SNP136

SNP137

SNP138

SNP139

 rs141122119

 5:83258556

 A/G

AA/AG/GG

258

I/V

 363/17/0

 2.28

1.10/2.04

 0.82/

0.37

 rs3734091

 5:83204915

 G/T

 rs371824973

 5:83204910

 G/A

 rs767176080

 5:83204907

 C/T

rs1261641487

 5:83204906

 T/C

rs1013137284

 5:83204904

 T/A

 rs542187236

 5:83204901

 A/G

 rs574436773

 5:83204897

 A/T

AA/AT/TT AA/AG/GG TT/TA/AA TT/TC/CC CC/CT/TT GG/GA/AA GG/GT/TT

241 242 243 244 244 245 247

A/S

 296/46/38

 2.09

 3.08

 0.77

0.97/2.79

 1.49/

1.20 1070

G/E

 380/0/0

S/F

 376/6/0

 2.27

 2.27

 1.09/

/

—

1.09/1.71

 0.34/

0.31

S/P

 380/0/0

L/H

 374/6/0

 2.26

 2.27

 1.09/

/

—

1.09/3.00

 0.56/

0.10

D/G

 380/0/0

T/S

 376/4/0

 2.27

 2.27

 1.09/

/

—

1.09/1.96

 1.19/

0.57

 rs762812825

 5:83204882

 G/A

 rs140579916

 5:83204874

 C/A

 rs368106955

 5:83204867

 G/A

 rs748307585

 5:83204852

 G/A

 rs774555675

 5:83204850

 G/A

rs866477694

 5:83204848

 C/A

rs1347664669

 5:83204847

 A/G

rs908326126

 5:83204840

 A/G

rs749647860

 5:83204828

 T/G

rs1348464342

 5:83204825

 A/G

AA/AG/GG TT/TG/GG AA/AG/GG AA/AG/GG CC/CA/AA GG/GA/AA GG/GA/AA GG/GA/AA CC/CA/AA GG/GA/AA

217 218 222 224 224 225 226 231 233 236

E/K

 357/21/2

 2.26

 2.23

 0.26

1.08/2.43

 1.33/

0.78

T/N

 342/38/0

 2.26

1.07/2.39

1.23/2.27

 1.09

 0.46

E/K

 380/0/0

D/N

 285/86/9

 2.29

1.09/2.20

2.27

 1.09/

/

—

1.09/2.37

 1.12

 0.77

*X-Ray Repair Cross Complementing 4 (XRCC4) Genetic Single Nucleotide Polymorphisms…*

R/Q

 348/32/0

 2.28

1.10/2.11

 0.97/

0.39

D/E

 380/0/0

D/G

 354/26/0

 2.26

 2.27

 1.09/

/

—

1.09/2.38

 1.12/

0.58

T/A

 380/0/0

C/G

 358/22/0

 2.27

 2.27

 1.09/

/

—

1.08/2.22

 1.23/

0.84

*DOI: http://dx.doi.org/10.5772/intechopen.88752*

I/V

 375/5/0

 2.27

1.09/2.46

 0.94/

0.69

rs780330653

 5:83204823

 C/A

rs969467594

 5:83204820

 C/G

 **SNP ID**

 **Chr: bp**

 **Alleles**

**Genotypes**

**Amino acid**

**Amino acid**

**Nxx/xy/yyc**

**Adducts (mean**

 **SD)**

*P***d**

**change**

**position**

215 216

A/E

 307/61/12

 2.24

 2.06

 0.72

1.08/2.46

 1.19/

0.28

T/S

 380/0/0

2.27

 1.09/

/

—

**(xx/xy/yy)b**

CC//CG/GG

CC/CA/AA

**(x/y)a**

*Aflatoxin B1 Occurrence, Detection and Toxicological Effects*


#### *X-Ray Repair Cross Complementing 4 (XRCC4) Genetic Single Nucleotide Polymorphisms… DOI: http://dx.doi.org/10.5772/intechopen.88752*

**No.**

**162**

SNP100

SNP101

SNP102

SNP103

SNP104

SNP105

SNP106

SNP107

SNP108

SNP109

SNP110

SNP111

SNP112

SNP113

SNP114

SNP115

SNP116

SNP117

SNP118

SNP119

rs746407658

 5:83204816

 G/A

rs1298401873

 5:83203706

 G/A

rs1276157833

 5:83203701

 A/G

rs201604424

 5:83203698

 A/G

rs1224705261

 5:83203694

 A/G

AA/AG/GG AA/AG/GG AA/AG/GG GG/GA/AA GG/GA/AA

209 210 211 213 214

E/K

 291/79/10

 2.30

1.10/2.22

1.09/1.72

 072

 0.23

G/R

 359/21/0

 2.29

1.10/1.93

 0.87/

0.14

Q/R

 371/9/0

 2.28

1.08/1.82

 1.17/

0.22

K/R

 300/72/8

 2.29

1.11/2.15

0.97/2.77

 1.03

 0.26

I/V

 380/0/0

 rs777199609

 5:83203691

 G/C

 rs778723397

 5:83203683

 G/A

 rs766287987

 5:83203678

 A/T

rs1263079073

 5:83203676

 C/G

rs1458486332

 5:83203659

 A/G

rs764109844

 5:83203654

 T/G

rs1173748737

 5:83203653

 A/G

 rs763186148

 5:83203652

 C/T

CC/CT/TT AA/AG/GG TT/TG/GG AA/AG/GG CC//CG/GG

AA/AT/TT GG/GA/AA GG/GC/CC

195 195 195 197 203 203 205 208

D/H

 292/86/2

 2.22

 2.86

2.27

 1.09/

/

—

 1.40

1.05/2.43

 1.18/

0.20

R/Q

 365/15/0

 2.29

1.09/1.87

 0.97/

0.15

Q/H

 357/16/7

 2.11

0.89/4.15

0.77/5.78

 1.17 1.05 1059

Q/E

 355/25/

2.28

1.09/2.18

 1.09/

0.69

K/R

 380/0/0

H/Q

 380/0/0

H/R

 380/0/0

H/Y

 328/50/2

 2.28

1.11/2.23

2.27

2.27

2.27

 1.09/

/

—

 1.09/

/

—

 1.09/

/

—

0.97/2.18

 0.47

 0.95

 rs775587299

 5:83203644

 G/T

 rs770217916

 5:83203641

 T/A

rs1482288279

 5:83203635

 C/T

rs1018879495

 5:83203623

 A/T

rs1199884817

 5:83203611

 T/C

rs1379408635

 5:83203610

 A/T

 rs777195630

 5:83203607

 T/C

 **SNP ID**

 **Chr: bp**

 **Alleles**

**Genotypes**

**Amino acid**

**Amino acid**

**Nxx/xy/yyc**

**Adducts (mean**

 **SD)**

*P***d**

**change**

**position**

180 181 181 185 189 191 192

R/I

 351/24/5

 2.28

 1.94

 0.64

1.09/2.24

 1.16/

0.79

I/N

 341/38/1

 2.28

1.10/2.10

0.90/4.31b

 0.11

*Aflatoxin B1 Occurrence, Detection and Toxicological Effects*

T/I

 380/0/0

N/I

 366/14/0

 2.27

 2.27

 1.09/

/

—

1.09/2.18

 0.94/

0.76

I/T

 345/35/

2.27

1.08/2.26

 1.20/

0.96

I/F

 338/41/1

 2.26

1.07/2.37

1.22/2.98e

0.67

F/L

 354/26/0

 2.22

1.04/2.89

 1.51/

0.04

**(xx/xy/yy)b**

TT/TC/CC AA/AT/TT TT/TC/CC AA/AT/TT CC/CT/TT TT/TA/AA GG/GT/TT


*dP values are calculated using t-test or one-way analysis of variance.*

*eSD is not determined.*

*\**

 *SD for genotype yy is not determined and P-value is used for genotypes xx and xy.*

#### **Table 2.**

*The association between SNPs in the coding region of XRCC4 and AFB1-DNA adducts in tissues with hepatocellular carcinoma.*

**Figure 1.**

**Figure 2.**

**165**

*likelihood ratio test.*

*XRCC4 GSNPs significantly correlating with the overall survival (OS) of hepatocellular carcinoma (HCC).*

*X-Ray Repair Cross Complementing 4 (XRCC4) Genetic Single Nucleotide Polymorphisms*

*DOI: http://dx.doi.org/10.5772/intechopen.88752*

*sided log-rank tests. The relative hazard ratio (HR) values for genotypes were calculated using multivariable Cox regression models (with all significant variables) based on forward-step method with likelihood ratio test.*

*XRCC4 GSNPs significantly correlating with the disease recurrence-free survival (RFS) of hepatocellular carcinoma (HCC). Cumulative hazard function was plotted by Kaplan-Meier methodology, and*

*calculated with two-sided log-rank tests. The relative hazard ratio (HR) values for genotypes were calculated using multivariable Cox regression models (with all significant variables) based on forward-step method with*

P *value was calculated with two-*

*…*

P *value was*

*Cumulative hazard function was plotted by Kaplan-Meier methodology, and*

*X-Ray Repair Cross Complementing 4 (XRCC4) Genetic Single Nucleotide Polymorphisms… DOI: http://dx.doi.org/10.5772/intechopen.88752*

**Figure 1.**

*XRCC4 GSNPs significantly correlating with the overall survival (OS) of hepatocellular carcinoma (HCC). Cumulative hazard function was plotted by Kaplan-Meier methodology, and* P *value was calculated with twosided log-rank tests. The relative hazard ratio (HR) values for genotypes were calculated using multivariable Cox regression models (with all significant variables) based on forward-step method with likelihood ratio test.*

#### **Figure 2.**

*XRCC4 GSNPs significantly correlating with the disease recurrence-free survival (RFS) of hepatocellular carcinoma (HCC). Cumulative hazard function was plotted by Kaplan-Meier methodology, and* P *value was calculated with two-sided log-rank tests. The relative hazard ratio (HR) values for genotypes were calculated using multivariable Cox regression models (with all significant variables) based on forward-step method with likelihood ratio test.*

**No.**

**164**

SNP140

SNP141

SNP142

SNP143

*ax/y represents wild-type allele/ variant type allele.*

*bxx/xy/yy represents wild-type* 

*c*

*Nxx/xy/yy represents the number of subjects with xx genotype, the number of subjects with xy genotype, and the number of subjects with yy genotype.*

*dP values are calculated using t-test or one-way analysis of variance.*

*eSD is not determined.* *\* SD for genotype yy is not determined and P-value is used for genotypes xx and xy.*

**Table 2.** *The association*

 *between SNPs in the coding region of XRCC4 and AFB1-DNA*

 *adducts in tissues with* 

*hepatocellular*

 *carcinoma.*

rs141304949

 5:83353238

 T/C *homozygote/heterozygote/variant-type*

 *homozygote.*

rs148273490

 5:83353231

 G/C

 rs61749611

 5:83353207

 A/C

 rs138837678

 5:83258665

 A/C

 **SNP ID**

 **Chr: bp**

 **Alleles**

**Genotypes**

**Amino acid**

**Amino acid**

**Nxx/xy/yyc**

**Adducts (mean**

 **SD)**

*P***d**

**change**

**position**

294 324 332 334

I/T

 344/35/1

 2.26

1.07/2.39

1.26/1.37e

0.58

*Aflatoxin B1 Occurrence, Detection and Toxicological Effects*

D/H

 356/24/0

 2.29

1.10/2.02

 0.86/

0.25

N/H

 348/30/2

 2.27

1.09/2.21

1.14/2.53

 0.16

 0.90

Q/P

 372/8/0

 2.28

1.09/1.98

 0.79/

0.45

**(xx/xy/yy)b**

AA/AC/CC AA/AC/CC GG/GC/CC

TT/TC/CC

genotypes (XX genotypes), the mutant genotypes (including XY and YY genotypes) of rs28383151, rs766287987, and rs3734091 polymorphisms increased cumulative hazard for OS [HR = 1.31 (1.04–1.67), 1.72 (1.09–2.71), and 1.42 (1.10–1.82), respectively] (**Figure 1**). For RFS, the corresponding hazard values were 3.31 (2.33– 4.69) for rs28383151, 1.85 (1.03–3.67) for rs766287987, and 4.45 (3.13–6.34) for rs3734091, respectively (**Figure 2**).

gene mutations [23, 24, 26, 27]. In our study, we tested the genotypic distributions of all known GSNPs in the coding region of XRCC4 in liver tumor tissues. Five positive GSNPs were identified, and they result in the change of amino acid D to Y at codon 38 for rs1237462915, A to T at codon 56 for rs28383151, I to T at codon 127 for rs762419679, Q to H at codon 203 for rs766287987, and A to S at codon 247 for rs3734091, respectively. Although evidence that several other GSNPs, including rs761695470, rs758779099, rs144653114, rs1277864722, and rs777195630, increased the amounts of AFB1-DNA adducts was not statistically significant according to our defined threshold value, their effects should not be neglected because small-size

*X-Ray Repair Cross Complementing 4 (XRCC4) Genetic Single Nucleotide Polymorphisms…*

Because the toxic effects of AFB1 also modify the prognosis of patients with HCC [26, 27, 33, 52, 53], we accomplished patients'survival analyses on the basis of the cumulative risk models and found only rs28383151, rs766287987, and rs3734091 polymorphisms shortened HCC cases' OS and RFS. Supporting our findings, several previous reports have proved that XRCC4 GSNPs can alter the levels of XRCC4 mRNA and protein expression and dysregulation of XRCC4 expression increasing the amount of AFB1-DNA adducts and mutative risk of TP53 gene [23, 24, 26, 27]. To conclude, this study is the first report investigating the modified function of XRCC4 GSNPs on AFB1's hepatic toxicity. Our findings suggest that the GSNPs in the coding regions of XRCC4 gene, like rs1237462915, rs28383151, rs762419679, rs766287987, and rs3734091, may alter the DNA repair capacity of DNA damage induced by AFB1. If these individuals with mutant genotypes of these GSNPs decrease their exposure to AFB1, they will be free from toxic effects of AFB1 on hepatic damage. Several limitations should be focused for our study. First, relatively small-size samples may underestimate the effects of XRCC4 GSNPs on AFB1 hepatic toxicity. Second, the hospital-based design may result in selective bias. Third, we only accomplished the cumulative risk analyses but not the cumulative survival analyses. Finally, we did not finish functional and mechanical analyses. Thus, XRCC4 GSNPs may be valuable biomarkers for predicting the toxic effects of AFB1 on the liver once the present findings were proved by larger samples and toxic

We thank Dr. Qiu-Xiang Liang, Dr. Yun Yi, Dr. Yun Xia, Dr. Yong-Zhi Huang,

The authors declare no competing financial interests. This study was supported

in part by the National Natural Science Foundation of China (Nos. 81860489, 81760502, 81572353, and 81660495), the Natural Science Foundation of Guangxi (Nos. 2018GXNSFAA281043, 2017GXNSFAA198002, and 2017GXNSFGA198002), Research Program of Guangxi "Zhouyue Scholar" (No. 2017-38), Research Program of Guangxi Specially Invited Expert (No. 2017-6th), "12th Five-Year" Planning Program of Guangxi Education Science (No. 2015C397), Innovative Program of Guangxi Graduate Education (No. JGY2015139), Research Program of Guangxi Clinic Research Center of Hepatobiliary Diseases (No. AD17129025), and Open

and Dr. Yuan-Feng Zhou for sample collection and management and Dr. Hua Huang for molecular biochemical technique. We also thank all members of the Department of Medical Test and Infective Control, Affiliated Hospital of Youjiang

Medical University for Nationalities for their help.

**Conflicts of interest and source of funding**

samples may underestimate values.

*DOI: http://dx.doi.org/10.5772/intechopen.88752*

function analyses.

**167**

**Acknowledgements**

## **4. Discussion**

In this study, we investigated the association between the GSNPs in the coding regions of XRCC4 gene and the toxic effects of AFB1 on the liver. We found that five XRCC4 GSNPs, including rs1237462915 (at codon 38), rs28383151 (at codon 56), rs762419679 (at codon 127), rs766287987 (at codon 203), and rs3734091 (at codon 247), significantly increased the amount of AFB1-DNA adducts in tissues with HCC (2.07–2.15 μmol/mol DNA for XY genotypes and 2.64–4.33 μmol/mol DNA for YY genotypes, respectively) and progressed the cumulative hazard of AFB1 hepatic toxicity.

AFB1 acts as a type of human chemical toxicant, and the toxic effects of this toxicant are characterized by organophilism (mainly causing hepatic damage), genic toxicity (mainly inducing DNA damages such as hotspot mutation at codon 249 of TP53 gene, AFB1-DNA adduct formation, and so on), and carcinogenicity (mainly resulting in HCC) [6–8]. Among the hepatic toxicity of AFB1, the formation of AFB1-DNA adducts in hepatic cells is a key step during the metabolism of this toxicant [9–14]. Evidence from molecular epidemiological studies and clinical studies has proved that the levels of AFB1-DNA adducts in the hepatic tissues are positively associated with the levels and time of AFB1 exposure [3, 24, 26–44]. This is indicative of AFB1-DNA adduct acting as the biomarker for AFB1's toxic capacity in the liver. In this study, AFB1-DNA adduct in the tumor tissues with HCC was used to evaluate hepatic toxicity related to AFB1, mainly because normal liver tissue samples cannot be obtained. Our results exhibited HCC tumor samples from high AFB1 exposure areas have an average adduct amount of 2.27 1.09 μmol/mol DNA. Supporting our findings, several studies from high AFB1 exposure areas Nanning and Tiandong, China, have also shown the similar level of DNA adducts [4, 5, 26, 27, 37, 39, 45]. Taken together, the amount of AFB1-DNA adducts should be able to reflect the hepatic toxic potential of AFB1.

XRCC4, a key gene in the V(D)J recombination repair pathway, is located at 5q14.2 and consists of 13 exons (PubMed). Normally, XRCC4 is mainly expressed in genital meatus, alimentary tract, and lymphoid tissue; however, its expression will noticeably increase in other tissues such as the skin and liver under the condition of in vitro and in vivo injuries. This gene's encoding protein plays a vital role in both NHEJ and the completion of V(D)J recombination via acting as a scaffold protein for DNA ligase IV and DNA-PK in the repair of DNA DSBs [15, 19]. Mutations in XRCC4, including GSNPs and other non-GSNPs variants, can cause endocrine dysfunction, microcephaly, short stature, and diseases [16, 21]. With the development of human Geno projects, more than 1000 GSNPs are identified. Among these GSNPs, we focused on genetic alterations in the coding regions of XRCC4, mainly because they will result in missense mutations and ultimately cause the structure damage and function deficiency of XRCC4 protein. Molecular epidemiological studies have displayed that the GSNPs in the XRCC4 genes can increase DNA repair capacity and increase the risk of some tumors such as lung cancer, colon cancer, HCC, and so on [21, 46–51]. Evidence from in vitro and in vivo studies has also proved that XRCC4 GSNPs increase the amount of DNA damage and induce more

#### *X-Ray Repair Cross Complementing 4 (XRCC4) Genetic Single Nucleotide Polymorphisms… DOI: http://dx.doi.org/10.5772/intechopen.88752*

gene mutations [23, 24, 26, 27]. In our study, we tested the genotypic distributions of all known GSNPs in the coding region of XRCC4 in liver tumor tissues. Five positive GSNPs were identified, and they result in the change of amino acid D to Y at codon 38 for rs1237462915, A to T at codon 56 for rs28383151, I to T at codon 127 for rs762419679, Q to H at codon 203 for rs766287987, and A to S at codon 247 for rs3734091, respectively. Although evidence that several other GSNPs, including rs761695470, rs758779099, rs144653114, rs1277864722, and rs777195630, increased the amounts of AFB1-DNA adducts was not statistically significant according to our defined threshold value, their effects should not be neglected because small-size samples may underestimate values.

Because the toxic effects of AFB1 also modify the prognosis of patients with HCC [26, 27, 33, 52, 53], we accomplished patients'survival analyses on the basis of the cumulative risk models and found only rs28383151, rs766287987, and rs3734091 polymorphisms shortened HCC cases' OS and RFS. Supporting our findings, several previous reports have proved that XRCC4 GSNPs can alter the levels of XRCC4 mRNA and protein expression and dysregulation of XRCC4 expression increasing the amount of AFB1-DNA adducts and mutative risk of TP53 gene [23, 24, 26, 27].

To conclude, this study is the first report investigating the modified function of XRCC4 GSNPs on AFB1's hepatic toxicity. Our findings suggest that the GSNPs in the coding regions of XRCC4 gene, like rs1237462915, rs28383151, rs762419679, rs766287987, and rs3734091, may alter the DNA repair capacity of DNA damage induced by AFB1. If these individuals with mutant genotypes of these GSNPs decrease their exposure to AFB1, they will be free from toxic effects of AFB1 on hepatic damage. Several limitations should be focused for our study. First, relatively small-size samples may underestimate the effects of XRCC4 GSNPs on AFB1 hepatic toxicity. Second, the hospital-based design may result in selective bias. Third, we only accomplished the cumulative risk analyses but not the cumulative survival analyses. Finally, we did not finish functional and mechanical analyses. Thus, XRCC4 GSNPs may be valuable biomarkers for predicting the toxic effects of AFB1 on the liver once the present findings were proved by larger samples and toxic function analyses.

### **Acknowledgements**

genotypes (XX genotypes), the mutant genotypes (including XY and YY genotypes) of rs28383151, rs766287987, and rs3734091 polymorphisms increased cumulative hazard for OS [HR = 1.31 (1.04–1.67), 1.72 (1.09–2.71), and 1.42 (1.10–1.82), respectively] (**Figure 1**). For RFS, the corresponding hazard values were 3.31 (2.33– 4.69) for rs28383151, 1.85 (1.03–3.67) for rs766287987, and 4.45 (3.13–6.34) for

In this study, we investigated the association between the GSNPs in the coding regions of XRCC4 gene and the toxic effects of AFB1 on the liver. We found that five XRCC4 GSNPs, including rs1237462915 (at codon 38), rs28383151 (at codon 56), rs762419679 (at codon 127), rs766287987 (at codon 203), and rs3734091 (at codon 247), significantly increased the amount of AFB1-DNA adducts in tissues with HCC (2.07–2.15 μmol/mol DNA for XY genotypes and 2.64–4.33 μmol/mol DNA for YY genotypes, respectively) and progressed the cumulative hazard of

AFB1 acts as a type of human chemical toxicant, and the toxic effects of this toxicant are characterized by organophilism (mainly causing hepatic damage), genic toxicity (mainly inducing DNA damages such as hotspot mutation at codon 249 of TP53 gene, AFB1-DNA adduct formation, and so on), and carcinogenicity (mainly resulting in HCC) [6–8]. Among the hepatic toxicity of AFB1, the formation of AFB1-DNA adducts in hepatic cells is a key step during the metabolism of this toxicant [9–14]. Evidence from molecular epidemiological studies and clinical studies has proved that the levels of AFB1-DNA adducts in the hepatic tissues are positively associated with the levels and time of AFB1 exposure [3, 24, 26–44]. This is indicative of AFB1-DNA adduct acting as the biomarker for AFB1's toxic capacity in the liver. In this study, AFB1-DNA adduct in the tumor tissues with HCC was used to evaluate hepatic toxicity related to AFB1, mainly because normal liver tissue samples cannot be obtained. Our results exhibited HCC tumor samples from high AFB1 exposure areas have an average adduct amount of 2.27 1.09 μmol/mol DNA. Supporting our findings, several studies from high AFB1 exposure areas Nanning and Tiandong, China, have also shown the similar level of DNA adducts [4, 5, 26, 27, 37, 39, 45]. Taken together, the amount of AFB1-DNA adducts should be able

XRCC4, a key gene in the V(D)J recombination repair pathway, is located at 5q14.2 and consists of 13 exons (PubMed). Normally, XRCC4 is mainly expressed in genital meatus, alimentary tract, and lymphoid tissue; however, its expression will noticeably increase in other tissues such as the skin and liver under the condition of in vitro and in vivo injuries. This gene's encoding protein plays a vital role in both NHEJ and the completion of V(D)J recombination via acting as a scaffold protein for DNA ligase IV and DNA-PK in the repair of DNA DSBs [15, 19]. Mutations in XRCC4, including GSNPs and other non-GSNPs variants, can cause endocrine dysfunction, microcephaly, short stature, and diseases [16, 21]. With the development of human Geno projects, more than 1000 GSNPs are identified. Among these GSNPs, we focused on genetic alterations in the coding regions of XRCC4, mainly because they will result in missense mutations and ultimately cause the structure damage and function deficiency of XRCC4 protein. Molecular epidemiological studies have displayed that the GSNPs in the XRCC4 genes can increase DNA repair capacity and increase the risk of some tumors such as lung cancer, colon cancer, HCC, and so on [21, 46–51]. Evidence from in vitro and in vivo studies has also proved that XRCC4 GSNPs increase the amount of DNA damage and induce more

rs3734091, respectively (**Figure 2**).

*Aflatoxin B1 Occurrence, Detection and Toxicological Effects*

**4. Discussion**

AFB1 hepatic toxicity.

**166**

to reflect the hepatic toxic potential of AFB1.

We thank Dr. Qiu-Xiang Liang, Dr. Yun Yi, Dr. Yun Xia, Dr. Yong-Zhi Huang, and Dr. Yuan-Feng Zhou for sample collection and management and Dr. Hua Huang for molecular biochemical technique. We also thank all members of the Department of Medical Test and Infective Control, Affiliated Hospital of Youjiang Medical University for Nationalities for their help.

## **Conflicts of interest and source of funding**

The authors declare no competing financial interests. This study was supported in part by the National Natural Science Foundation of China (Nos. 81860489, 81760502, 81572353, and 81660495), the Natural Science Foundation of Guangxi (Nos. 2018GXNSFAA281043, 2017GXNSFAA198002, and 2017GXNSFGA198002), Research Program of Guangxi "Zhouyue Scholar" (No. 2017-38), Research Program of Guangxi Specially Invited Expert (No. 2017-6th), "12th Five-Year" Planning Program of Guangxi Education Science (No. 2015C397), Innovative Program of Guangxi Graduate Education (No. JGY2015139), Research Program of Guangxi Clinic Research Center of Hepatobiliary Diseases (No. AD17129025), and Open

Research Program from Molecular Immunity Study Room Involving in Acute and Severe Diseases in Guangxi Colleges and Universities (Nos. kfkt20160062 and kfkt20160063).

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